Small-molecule Inhibition of the Interactions of the Urokinase Receptor: A Targe

项目摘要/摘要 尿激酶受体(UPAR)是一种与gpi结合的蛋白，可作为丝氨酸蛋白酶的受体。 尿激酶(UPA)。一旦与其受体对接，uPA就被激活，这是一种导致细胞周围蛋白分解的事件 细胞外基质的降解，这是肿瘤和血管系统之间的主要屏障。但现在是时候了 人们普遍认为，虽然uPAR不能发出信号，但uPAR/uPA复合体相互作用并激活 许多细胞表面受体，如整合素、受体酪氨酸激酶和GPCRs。UPAR/UPA 相互作用与肿瘤侵袭和转移的几乎每一步都有关，因此是一种 非常适合作为抗癌治疗药物开发的靶点。之前的努力集中在 用小分子物质抑制uPA丝氨酸蛋白酶活性。但这种策略忽略了 UPAR/uPA与细胞信号蛋白的复合体，不依赖于uPA的蛋白分解活性。在这 在应用程序中，我们遵循了一种独特的方法，即通过使用 小分子，期望这些分子将表现出阻断蛋白分解的双重效果 和信号。我们最初的努力取得了很高的回报。一种基于结构的初步计算 筛选得到了8个活性化合物。基于细胞培养的研究表明，这些化合物中有许多 阻断MDA-MB-231肿瘤细胞的黏附、迁移和侵袭。化合物也被发现抑制肺 H1299肿瘤细胞增殖。生化分析表明，这些化合物也能阻断整合素。 结合uPAR/uPA复合体。我们在这项资助申请中的目标是优化药物动力学 其中三种化合物在体内阻断肿瘤侵袭和转移的性质和效力。到那时候 一项涉及计算生物学、合成化学、生物物理学的多学科研究计划 接下来将学习化学、结构生物学和细胞生物学。第一个目标将包括创建一个虚拟的 基于我们的先导分子的化合物组合库，根据以下条件对这些化合物进行排序 预测效力，并预测最有效化合物的药代动力学特性。第二个目标 将涉及最有希望的化合物的化学合成，然后评估它们的 用荧光偏振法测定活性。最有效的化合物进一步被表征为 等温滴定量热法。然后我们在复数中求解这些化合物的三维结构 用X-射线衍射法与尿激活酶受体结合。最后，第三个目标将包括评估细胞 最具选择性和最有效的抑制剂在MDA-MB-231和其他肿瘤细胞系中的有效性，并进行 初步的体内剂量研究，为未来的小鼠异种移植研究奠定基础。

PROJECT SUMMARY/ABSTRACT The urokinase receptor (uPAR) is a GPI-anchored protein that serves as a receptor to the serine protease urokinase (uPA). Upon docking to its receptor, uPA is activated, an event that leads to pericellular proteolysis and degradation of the extracellular matrix, a primary barrier between tumor and vasculature. But it is now widely recognized that while uPAR is not capable of signaling, the uPAR/uPA complex interacts and activates a number of cell surface receptors such as integrins, receptor tyrosine kinases, and GPCRs. The uPAR/uPA interaction has been implicated with nearly every step of tumor invasion and metastasis and is therefore a highly suitable target for the development of anti-cancer therapeutics. Previous efforts have concentrated on inhibiting the serine protease activity of uPA with small molecules. But this strategy neglects interactions of the uPAR/uPA complex with cell signaling proteins that are independent of the proteolytic activity of uPA. In this application, we follow a unique approach that seeks to target the uPAR/uPA protein-protein interaction using small molecules with the expectation that these molecules will exhibit the dual effect of blocking proteolysis and signaling. Our initial efforts have been highly rewarding. A preliminary structure-based computational screen has led to 8 active compounds. Cell culture-based studies reveal that a number of these compounds block MDA-MB-231 tumor cell adhesion, migration, and invasion. Compounds were also found to inhibit lung H1299 tumor cell proliferation. Biochemical analyses reveal that that these compounds also block integrin binding to the uPAR/uPA complex. Our objective in this grant application is to optimize the pharmacokinetic properties and potency of three of these compounds to block tumor invasion and metastasis in vivo. To that end, a multidisciplinary research program involving computational biology, synthetic chemistry, biophysical chemistry, structural biology, and cell biology will be followed. The first aim will consist of creating a virtual combinatorial library of compounds based on our lead molecules, ranking these compounds based on predicted potency, and predicting pharmacokinetic properties of the most potent compounds. The second aim will involve the chemical synthesis of the most promising compounds, followed by an assessment of their activity using a fluorescence polarization assay. The most potent compounds are further characterized with isothermal titration calorimetry. We then solve the three-dimensional structure of these compounds in complex with the urokinase receptor using x-ray diffraction. Finally, the third aim will consist of assessing the cellular efficacy of the most selective and potent inhibitors in MDA-MB-231 and other tumor cell lines, and to perform preliminary in vivo dosage studies to set the stage for future studies in mice xenografts.