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

描述（由申请人提供）：炎性巨噬细胞在许多疾病中发挥重要作用，经常涉及炎症从慢性到急性状态的转变。在结核病中，当肉芽肿开始破裂，最终形成活动性疾病时，就会出现这种情况。活动性疾病状态已被证明与基质金属蛋白酶（MMP）表达水平升高相关。在小鼠模型中，我们可以利用分枝杆菌脂质海藻糖二mycolate （TDM）诱导巨噬细胞采用类似的破坏性侵袭性表型，该表型也以MMP活性水平升高为标志。我们建议利用这个模型来开发一个HTS平台来识别炎症巨噬细胞的抑制剂。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源性因子。这些因素的确定将使吞噬细胞活化步骤的优化和标准化成为可能。这将通过重组蛋白或通过对肉芽肿条件培养基中活性蛋白的定量来实现。目标2。开发HTS平台筛选mmp依赖性组织破坏的小分子抑制剂。我们将在多孔板试验中使用荧光MMP或组织蛋白酶底物来筛选抑制巨噬细胞激活诱导或下游后果的抑制剂。目标3。在主屏幕中获得的“命中”的验证和功能分类。我们将开发二级筛选，以促进假阳性的快速识别，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。该项目的目标是筛选我们自己的内部库（150,000种化合物），并利用这些数据扩展项目并吸引工业合作伙伴。

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.