Novel Small-Molecule Inhibitors of the Tie2/Angiopoietin Interactions and Signali

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DESCRIPTION (provided by applicant): The goal of this study is to develop a robust and sensitive assay and to implement it for the high throughput screening (HTS) of chemical libraries for compounds that block the Tie2/Angiopoietin interactions and signaling. The Tie2 receptor tyrosine kinase and its angiopoietin ligands regulate both developmental and tumor-induced angiogenesis and, therefore, our proposed research will facilitate the development of novel anti-tumor strategies based on the targeted disruption of tumor-induced blood vessel formation. Our preliminary structural, biophysical and biochemical characterization of the angiopoietins, Tie2, and of their interactions, suggests that small-molecules would be able to disrupt the Tie2/Ang complex formation and the initiation of Tie2-mediated signaling. Furthermore, we are currently developing a Homogeneous Time Resolved Fluorescence (HTRF) ligand-receptor interactions assay, which we have documented is suitable for implementation in a high-throughput screen format. We have also identified suitable secondary- and counter- screening assays for "hit" validation and prioritization. We propose to further optimize the primary HTRF assay, configure it in HTS format and perform a validation screen of ~2700 selected compounds. Subsequently, we propose to implement this assay to screen a complete compound library for small molecules that disrupt respectively the Tie2/Ang1 and the Tie2/Ang2 complex formation. Finally, we will use a variety of in vitro and in vivo biochemical, biophysical, as well as cell- and animal-based assays, to validate the HTS hits and to characterize their biological properties, cytotoxicity and potential as anti-angiogenesis-based anti-cancer agents.