Development of a Cell-Based HTS for Inhibitors of Inflammatory Macrophages

描述(申请人提供)：炎性巨噬细胞在许多疾病中发挥重要作用，并经常与炎症从慢性状态向急性状态的转变有关。在结核病中，当肉芽肿开始破裂，最终导致活动性疾病时，就会发生这种情况。疾病的活跃状态已被证明与基质金属蛋白酶(MMP)的表达水平升高有关。利用小鼠模型，我们可以利用分枝杆菌脂质海藻糖二乙醇酸盐(TDM)来诱导巨噬细胞采用类似的破坏性、侵袭性表型，其特征也是基质金属蛋白酶活性水平升高。我们建议利用这一模型开发一个HTS平台来识别炎性巨噬细胞的抑制物。由于这是一种基于细胞的筛选，它将包括从吞噬细胞激活到细胞外基质降解的过程中的所有阶段。目的1：鉴定触发巨噬细胞破坏性侵袭的小鼠肉芽肿衍生因子。这些因素的识别将使吞噬细胞激活步骤的优化和标准化成为可能。这将通过重组蛋白或通过肉芽肿条件培养液中活性蛋白的量化来实现。目的2.建立一种用于筛选基质金属蛋白酶依赖的组织破坏的小分子抑制物的HTS平台。我们将在多孔板试验中使用荧光基质金属蛋白酶或组织蛋白酶底物来筛选阻断巨噬细胞激活的诱导或下游后果的抑制剂。目的3.对在主屏幕中获得的“命中”进行验证和功能分类。我们将开发二次筛选，以便于快速识别假阳性，并将活性化合物分类为影响激活、入侵或降解的不同功能类别。该项目的目标是筛选我们自己的库(15万种化合物)，并使用这些数据来扩大计划并吸引工业合作伙伴。 公共卫生相关性：炎性巨噬细胞在感染性和非感染性疾病的病理恶化中发挥关键作用。我们建议利用MTB细胞壁脂在小鼠巨噬细胞中诱导这种炎症状态的能力来开发基于细胞的HTS平台来筛选小分子抑制剂。这些抑制剂将是在动物模型中操纵炎症性疾病的有用探针，并将成为抗炎或调节对结核病等感染反应的药物的新候选药物。

DESCRIPTION (provided by applicant): Inflammatory macrophages play a significant role in many diseases and are frequently implicated in the transition of inflammation from a chronic to an acute state. In tuberculosis this occurs when the granuloma starts to breakdown, culminating in active disease. The active disease state has been shown to correlate with elevated levels of matrix metalloproteinase (MMP) expression. Using a murine model we can exploit the mycobacterial lipid trehalose dimycolate (TDM) to induce macrophages to adopt a similar destructive, invasive phenotype that is also marked by elevated levels of MMP activity. We propose exploiting this model to develop a HTS platform to identify inhibitors of inflammatory macrophages. As this is a cell-based screen it would incorporate all the stages in the process from activation of the phagocytes through to degradation of extracellular matrix. Aim 1: Identification of the murine granuloma-derived factors that trigger destructive invasion in macrophages. The identification of these factors will enable the optimization and standardization of the phagocyte activation step. This will be achieved either with recombinant proteins or through the quantification of the active proteins in the granuloma-conditioned medium. Aim 2. Development of a HTS platform to screen for small molecule inhibitors of MMP-dependent tissue destruction. We will use fluorogenic MMP or cathepsin substrates in a multwell plate assay to screen for inhibitors that block either the induction or the downstream consequences of macrophage activation. Aim 3. Validation and functional classification of the "hits" obtained in the primary screen. We will develop secondary screens to facilitate the rapid identification of false positives, and to categorize the active compounds into different functional classes that impact activation, or invasion, or degradation. The goal of the project is to screen our own in-house library (150,000 compounds) and use these data to expand the program and to attract an industrial partner. PUBLIC HEALTH RELEVANCE: Inflammatory macrophages play key roles in the exacerbation of pathology in both infectious and non- infectious diseases. We propose exploiting the ability of Mtb cell wall lipids to induce this inflammatory state in murine macrophages to develop a cell-based HTS platform to screen for small molecule inhibitors. Such inhibitors would be useful probes to manipulate inflammatory diseases in animal models and would be new candidates for drugs to combat inflammation or modulate the response to infections like tuberculosis.