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.
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.
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