HTS for bactericidal inhibitors of M. tuberculosis copper resistance mechanisms

描述（由申请人提供）：每年有140万人死于结核分枝杆菌（Mtb）感染，结核病（TB）被认为是一种大流行，越来越难以控制。结核病和艾滋病毒感染之间的致命协同作用以及耐药结核菌株的惊人数量威胁到我们控制结核病的能力，即使在发达国家也是如此。为了阻止和扭转这一发展趋势，需要能够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。我们最近发现铜稳态是结核分枝杆菌的一个重要毒力因子。我们可以证明，遗传扰动的Mtb铜稳态诱导铜超敏，这与强毒力缺陷有关。巨噬细胞利用铜离子杀死结核分枝杆菌，因此与铜依赖性先天免疫功能协同的化合物为治疗干扰创造了新的机会。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，用于鉴定铜促进药物，这些药物要么(i)抑制（ii）的单个成分，要么增加铜的摄取，从而压制结核分枝杆菌固有的铜抗性途径。在Aim 1中，我们将利用BSL2批准的Mtb H37Rv突变体优化并将HTS增铜药物筛选试验转移到机器人平台上。在目标2中，我们将开发和验证新的和更快速的二级分析，这将指导命中优先级。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生存能力。这种评估比传统的CFU计数快1000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1000个。设计各种检测方法的目的是测试确定的击中物是否作为杀菌或抑菌化合物，是否表现出对抗潜伏（不生长）结核分枝杆菌的活性，或是否与抗逆转录病毒药物相互作用。在这项应用的研究中，我们已经找到了两个证据

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).