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

描述(申请人提供)：本研究的目标是开发一种可靠和灵敏的分析方法，并将其用于高通量筛选(HTS)阻止Tie2/Angiopoietin相互作用和信号传递的化合物的化学文库。Tie2受体酪氨酸激酶及其血管生成素配体调节发育和肿瘤诱导的血管生成，因此，我们的研究将促进基于靶向干扰肿瘤诱导的血管形成的新的抗肿瘤策略的开发。我们对血管生成素Tie2及其相互作用的初步结构、生物物理和生化特征表明，小分子将能够破坏Tie2/Ang复合体的形成和Tie2介导的信号传导的启动。此外，我们目前正在开发一种均相时间分辨荧光(HTRF)配体-受体相互作用分析方法，我们已经证明这种方法适合在高通量屏幕格式中实现。我们还确定了用于“HIT”验证和优先排序的合适的二次和反筛选检测方法。我们建议进一步优化主要的HTRF分析，将其配置为HTS格式，并对约2700种选定的化合物进行验证筛选。随后，我们建议实施这一实验来筛选分别破坏Tie2/Ang1和Tie2/Ang2复合体形成的小分子的完整化合物文库。最后，我们将使用各种体外和体内生化、生物物理以及基于细胞和动物的分析方法来验证HTS HITS，并表征其生物学特性、细胞毒性和作为抗血管生成抗癌药物的潜力。

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.