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

描述（由申请方提供）：本研究的目的是开发一种稳健且灵敏的测定方法，并将其用于阻断Tie 2/血管生成素相互作用和信号传导的化合物的化学文库的高通量筛选（HTS）。Tie 2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，我们提出的研究将促进基于靶向破坏肿瘤诱导的血管形成的新型抗肿瘤策略的发展。我们对血管生成素Tie 2及其相互作用的初步结构、生物物理和生物化学表征表明，小分子能够破坏Tie 2/Ang复合物的形成和Tie 2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-受体相互作用测定法，我们已经证明该测定法适合于以高通量筛选形式实施。我们还确定了合适的二级和反筛选试验的“命中”验证和优先级。我们建议进一步优化主要HTRF测定，将其配置为HTS格式，并对约2700种选定化合物进行验证筛选。随后，我们建议实施该测定以筛选分别破坏Tie 2/Ang 1和Tie 2/Ang 2复合物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物化学，生物物理，以及细胞和动物为基础的测定，以验证HTS命中，并表征其生物学特性，细胞毒性和潜在的抗血管生成为基础的抗癌剂。

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.