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）在肿瘤的生长和血行播散中起重要作用，抑制血管生成作为限制肿瘤生长和扩散的新型有效治疗策略正日益受到重视。 内皮生长因子在血管生成中起主导作用，其中许多依赖于乙酰肝素的存在以结合其同源细胞表面受体，并随后启动导致内皮细胞增殖的下游信号级联。 我们推测，肝素的隔离将防止肝素依赖性生长因子与内皮细胞的结合，并抑制其促有丝分裂作用。 基于对硫酸乙酰肝素分子识别的结构特征的现有知识，我们已经构建了硫酸乙酰肝素结合的初步药效团，并建议筛选精心选择的化合物集中库。 我们将首先测试的假设，小分子拥有的药效团将结合硫酸乙酰肝素，使用肝素作为模型肝素化合物。 然后，我们将检查结合是否将表现在使用一小部分化合物抑制内皮生长因子活性。 利用一种新的染料置换分光光度法作为主要的高通量筛选已经产生了临时的“命中”，其中之一已被证明可以抑制FGF和VEGF诱导的血管生成活性在人脐内皮细胞。 将更详细地检查有前途的化合物与肝素的物理相互作用的细节。 进一步的生物活性将在鸡绒毛尿囊膜试验中确定。

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.