Novel High-throghput Assay:Angiogenesis Inhibitors (RMI)

描述（申请人提供）：肿瘤的血管形成（血管生成）在肿瘤的生长和血液播散中起着重要作用，抑制血管生成越来越被认为是限制肿瘤生长和扩散的一种新的有效治疗策略。内皮生长因子在血管生成中起主导作用，其中许多依赖于肝素的存在，以结合其同源细胞表面受体，并随后启动下游信号级联，导致内皮细胞增殖。我们假设肝素的隔离会阻止肝素依赖性生长因子与内皮细胞的结合并抑制其有丝分裂作用。基于对硫酸肝素分子识别的结构条件的现有知识，我们构建了硫酸肝素结合的初步药效团，并提出筛选一个精心选择的重点化合物库。我们将首先以肝素为模型肝素化合物，验证具有药效团的小分子与硫酸肝素结合的假设。然后，我们将检查这种结合是否会表现为使用一小部分化合物抑制内皮生长因子的活性。利用一种新型染料置换分光光度法作为主要的高通量筛选已经取得了初步的“成功”，其中一种已被证明可以抑制人类脐带内皮细胞中FGF和vegf诱导的血管生成活性。有前途的化合物将在与肝素物理相互作用的细节方面进行更详细的研究。进一步的生物活性将在鸡绒毛膜尿囊膜测定中确定。

DESCRIPTION (provided by applicant): The vascularization (angiogenesis) of tumors plays an important role in the growth and hematogenous dissemination of neoplasms, and the inhibition of angiogenesis is being increasingly appreciated as a novel and effective therapeutic strategy for limiting tumor growth and spread. Endothelial growth factors play a dominant role in angiogenesis, many of which are dependent on the presence of heparan for binding to their cognate cell-surface receptors and subsequent initiation of downstream signaling cascades that lead to endothelial cell proliferation. We hypothesize that the sequestration of heparan would prevent the binding of heparan-dependent growth factors to endothelial cells and inhibit their mitogenic effects. Based on existing knowledge on the structural requisites for the molecular recognition of heparan sulfate, we have constructed a preliminary pharmacophore for heparan sulfate binding, and propose to screen a carefully chosen focused library of compounds. We will first test the hypothesis that small molecules possessing the pharmacophore will bind heparan sulfate, using heparin as a model heparin compound. We will then examine if the binding will be manifested in inhibition of endothelial growth factor activity using a small subset of compounds. The utilization of a novel dye-displacement spectrophotometric assay as the primary high-throughput screen has already yielded provisional "hits", one of which has been shown to inhibit both FGF and VEGF-induced angiogenic activity in human umbilical endothelial cells. Promising compounds will be examined in greater detail with respect to details of the physical interactions with heparin. Further biological activity will be ascertained in chicken chorioallantoic membrane assays.