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起重要作用，并且经常参与炎症从慢性向急性状态的转变。在结核病中，当肉芽肿开始破裂时，这种情况发生，最终导致活动性疾病。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。使用小鼠模型，我们可以利用分枝杆菌脂质海藻糖二霉菌酸酯（TDM）诱导巨噬细胞采取类似的破坏性，侵入性表型，也标志着MMP活性水平升高。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的活化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。这些因素的鉴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定量肉芽肿条件培养基中的活性蛋白来实现。目标二。开发HTS平台以筛选MMP依赖性组织破坏的小分子抑制剂。我们将使用荧光基质金属蛋白酶或组织蛋白酶底物在multwell板测定，以筛选抑制剂，阻止诱导或下游的巨噬细胞活化的后果。目标3.对初步筛选中获得的“命中”进行验证和功能分类。我们将开发二次筛选，以促进假阳性的快速识别，并将活性化合物分类为影响激活或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内部库（150，000种化合物），并使用这些数据来扩展该计划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.