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

描述（由申请人提供）：尿激酶受体（uPAR）是一种gpi锚定蛋白，作为丝氨酸蛋白酶尿激酶（uPA）的受体。在与受体对接后，uPA被激活，这一事件导致细胞周围蛋白水解和细胞外基质的降解，细胞外基质是肿瘤和脉管系统之间的主要屏障。但现在人们普遍认识到，虽然uPAR不能发出信号，但uPAR/uPA复合物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gpcr。uPAR/uPA相互作用几乎与肿瘤侵袭和转移的每一步都有关系，因此是开发抗癌治疗药物的一个非常合适的靶点。以往的研究主要集中在用小分子抑制uPA的丝氨酸蛋白酶活性。但是这种策略忽略了uPAR/uPA复合物与独立于uPA蛋白水解活性的细胞信号蛋白的相互作用。在本应用中，我们采用了一种独特的方法，旨在利用小分子靶向uPAR/uPA蛋白-蛋白相互作用，期望这些分子能够表现出阻断蛋白水解和信号传导的双重作用。我们最初的努力得到了极大的回报。初步的基于结构的计算筛选得到了8种活性化合物。基于细胞培养的研究表明，许多这些化合物阻断MDA-MB-231肿瘤细胞的粘附、迁移和侵袭。化合物也被发现抑制肺H1299肿瘤细胞的增殖。生化分析表明，这些化合物也阻断整合素与uPAR/uPA复合物的结合。我们的目标是优化其中三种化合物的药代动力学特性和效力，以阻止肿瘤在体内的侵袭和转移。为此，一项涉及计算生物学、合成化学、生物物理化学、结构生物学和细胞生物学的多学科研究计划将紧随其后。第一个目标将包括基于我们的先导分子创建一个虚拟的化合物组合库，根据预测的效价对这些化合物进行排名，并预测最有效的化合物的药代动力学性质。第二个目标将涉及最有希望的化合物的化学合成，然后使用荧光偏振法评估其活性。最有效的化合物用等温滴定量热法进一步表征。然后我们用x射线衍射解决了这些化合物与尿激酶受体复合物的三维结构。最后，第三个目标将包括评估MDA-MB-231和其他肿瘤细胞系中最具选择性和最有效的抑制剂的细胞功效，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。公共卫生相关性：尿激酶受体是一种结合和激活多种蛋白质的细胞表面受体。除其他外，这些相互作用导致原发肿瘤和脉管系统之间的保护层退化，以及细胞的粘附和迁移。这些过程是癌症转移的标志。具有药物样特性的小分子可以阻止尿激酶受体与其结合伙伴相互作用，这可能会导致非常有效的治疗方法，以防止肿瘤转移。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The urokinase receptor is a cell surface receptor that binds and activates various proteins. These interactions lead, among other things, to the degradation of a protective layer between a primary tumor and the vasculature, as well as the adhesion and migration of cells. These processes are hallmarks of cancer metastasis. Small molecules with drug-like properties that prevent the urokinase receptor from interacting with its binding partners could lead to highly effective therapeutics to prevent tumor metastasis.