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

尿激酶受体相互作用的小分子抑制：一个目标

基本信息

批准号：
8035963
负责人：
Samy Meroueh
金额：
$ 24.8万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-15 至 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8035963
关键词：
Adhesions Affinity Antineoplastic Agents Applications Grants Binding Biochemical Biological Assay Blood - brain barrier anatomy Breast Caco-2 Cells Calorimetry Cell Adhesion Cell Culture Techniques Cell Line Cell Proliferation Cell Surface Proteins Cell Surface Receptors Cell surface Cells Cellular biology Chemicals Clinic Clinical Pharmacology Co-Immunoprecipitations Collaborations Colon Adenocarcinoma Complement Complex Computational Biology Core Facility Crystallization Cytochrome P450 Data Development Docking Drug Design Drug Kinetics Drug Transport Ensure Environment Evaluation Event Exhibits Extracellular Matrix Degradation Fluorescence Polarization Free Energy Future G protein coupled receptor kinase Generations Genomics Glioblastoma H1299 Head Health Human Indiana Integrin Binding Integrins Interdisciplinary Study Knowledge Lead Letters Libraries Ligands Literature Lung Malignant Neoplasms Mammary Neoplasms Mediating Metabolism Molecular Mus Neoplasm Metastasis Penetration Peptide Hydrolases Permeability Pharmaceutical Preparations Pharmacologic Substance Phosphorylation Plasminogen Positioning Attribute Primary Neoplasm Process Property Proteins Proteolysis Proteome Reporting Research Rewards Risk Role Screening procedure Serine Protease Serum Albumin Signal Pathway Signal Transduction Signaling Protein Site Solutions Staging Statistical Models Structure Surface Plasmon Resonance Synthesis Chemistry System Systems Development Testing Therapeutic Titrations Toxic effect Transmembrane Domain Tumor Cell Invasion Tumor Cell Line U118 Urokinase Urokinase Plasminogen Activator Receptor Validation Work Xenograft procedure angiogenesis anti-cancer therapeutic base biophysical chemistry cell motility chemical property chemical synthesis cheminformatics combinatorial crosslink design dosage drug discovery expectation experience fibrosarcoma forging high throughput screening in vivo inhibitor/antagonist innovation insight medical schools migration molecular dynamics neglect neoplastic cell prevent professor programs protein protein interaction receptor receptor binding small molecule small molecule libraries structural biology success three dimensional structure tumor tumor xenograft tyrosine receptor virtual

项目摘要

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.

描述（由申请人提供）：尿激酶受体（uPAR）是一种GPI锚定蛋白，充当丝氨酸蛋白酶尿激酶（uPA）的受体。一旦与其受体对接，uPA 就会被激活，这一事件会导致细胞周蛋白水解和细胞外基质（肿瘤和脉管系统之间的主要屏障）的降解。但现在人们普遍认识到，虽然 uPAR 不能发出信号，但 uPAR/uPA 复合物会相互作用并激活许多细胞表面受体，例如整合素、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有关，因此是开发抗癌疗法的非常合适的靶点。之前的努力主要集中在用小分子抑制uPA的丝氨酸蛋白酶活性。但这种策略忽略了 uPAR/uPA 复合物与细胞信号蛋白的相互作用，而细胞信号蛋白与 uPA 的蛋白水解活性无关。在此应用中，我们采用了一种独特的方法，旨在使用小分子靶向 uPAR/uPA 蛋白质-蛋白质相互作用，并期望这些分子将表现出阻断蛋白水解和信号转导的双重作用。我们最初的努力得到了丰厚的回报。基于结构的初步计算筛选已产生 8 种活性化合物。基于细胞培养的研究表明，许多此类化合物可阻断 MDA-MB-231 肿瘤细胞粘附、迁移和侵袭。还发现化合物可以抑制肺 H1299 肿瘤细胞增殖。生化分析表明，这些化合物还可阻断整合素与 uPAR/uPA 复合物的结合。我们在这项拨款申请中的目标是优化其中三种化合物的药代动力学特性和效力，以阻止体内肿瘤侵袭和转移。为此，将开展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。第一个目标包括根据我们的先导分子创建一个虚拟化合物组合库，根据预测的效力对这些化合物进行排名，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，然后使用荧光偏振测定评估其活性。最有效的化合物通过等温滴定量热法进一步表征。然后，我们使用 X 射线衍射解析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。公共健康相关性：尿激酶受体是一种细胞表面受体，可结合并激活各种蛋白质。这些相互作用尤其导致原发肿瘤和脉管系统之间的保护层的降解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，从而可以产生高效的治疗方法来预防肿瘤转移。