HTS for bactericidal inhibitors of M. tuberculosis copper resistance mechanisms

描述(申请人提供)：每年有140万人死于结核分枝杆菌(MTB)感染，结核病(TB)被认为是一种流行病，越来越难以控制。结核病和艾滋病毒感染之间致命的协同作用，以及数量惊人的耐药结核分枝杆菌菌株，威胁着我们控制结核病的能力，即使在发达国家也是如此。为了阻止和逆转这一发展，需要显著缩短和简化结核病治疗并使用新的药物靶点的新疗法。我们最近发现铜的动态平衡是结核分枝杆菌重要的毒力因子。我们可以证明，Mtb铜稳态的遗传扰动导致了铜超敏，这与一个强大的毒力缺陷有关。铜离子被巨噬细胞用来杀死结核分枝杆菌，因此与铜依赖的天然免疫功能协同的化合物为治疗干预创造了新的机会。在这个项目中，我们将开发一种强大的组合高通量筛选(HTS)方法来鉴定提高铜含量的药物，这些药物可以(I)抑制(Ii)的单个成分，或者增加铜的摄取，从而压倒Mtb固有的铜耐药途径。在目标1中，我们将优化HTS对铜促进药物的筛选试验，并将其转移到利用BSL2批准的Mtb H37Rv突变体的机器人平台。在目标2中，我们将开发和验证新的和更快速的二次分析，这将指导HIT优先顺序。特别是，我们将验证和利用基于流式细胞术的方法来评估药物暴露的无毒结核分枝杆菌细胞的活性。此评估比传统CFU计数快1,000倍，与HTS兼容，支持动态读出，每次读取和条件所需的单个细胞少于1,000个。各种检测方法旨在测试已识别的HITS是否作为杀菌或抑菌化合物，显示出对潜伏(不生长)的结核分枝杆菌的活性，或与抗逆转录病毒药物相互作用。在导致这一应用的研究中，我们已经确定了两个证据 以铜依赖的方式以Mtb为靶点的概念化合物，其体外治疗指数为&gt；20。在目标3中，我们将进行中试筛选以评估整体检测性能。在这一试点活动完成后，我们打算申请我们的筛选试验进入NIH分子文库探针生产中心网络(AIM 4)的快速通道。

DESCRIPTION (provided by applicant): With > 1.4 million people dying every year from Mycobacterium tuberculosis (Mtb) infection, tuberculosis (TB) is considered a pandemic, which is increasingly difficult to control. The deadly synergy between TB and HIV infection, and the alarming number of drug-resistant Mtb strains threaten our ability to control TB, even in developed countries. To halt and reverse this development, novel therapeutics that dramatically shorten and simplify TB treatment and that use new drug targets are needed. We recently discovered copper homeostasis as an important virulence factor of Mtb. We can demonstrate that genetic perturbation of Mtb copper homeostasis induces copper hypersensitivity which is associated with a strong virulence defect. Copper ions are employed by macrophages to kill Mtb hence compounds that synergize with copper dependent innate immune functions create novel opportunities for therapeutic interference. In this project we will develop a robust combinatorial high-throughput screening (HTS) assay for the identification of copper-boosting drugs that either (i) inhibit individual components of (ii) or increase copper uptake and thereby overwhelm the intrinsic copper resistance pathways of Mtb. In Aim 1, we will optimize and transfer an HTS screening assay for copper-boosting drugs to a robotic platform utilizing a BSL2 approved mutant of Mtb H37Rv. In Aim 2, we will develop and validate novel and more rapid secondary assays, which will guide hit prioritization. In particular, we will validate and utilize a flow cytometry-based approach to evaluate viability of drug exposed avirulent Mtb cells. This assessment is 1,000 times faster than traditional CFU counts, is HTS compatible, supports kinetic read-outs and requires less than 1,000 individual cells per read and condition. The various assays are designed to test whether the identified hits act as bactericidal or bacteriostatic compounds, exhibit activity against latent (non-growing) Mtb or interact with antiretroviral drugs. In research leading to this application, we have already identified two proof of concept compounds that target Mtb in a copper dependent manner and exhibit an in vitro therapeutic index of >20. In Aim 3, we will perform a pilot screen to evaluate overall assay performance. Upon completion of this pilot campaign, we intend to apply for Fast Track entry of our screening assay into the NIH Molecular Libraries Probe Production Centers Network (Aim 4).