Development of assays for HTS to identify inhibitors of a new PPI involved in cancer metastasis

摘要 乳腺癌是一个全球性问题，每年有130万新确诊病例，43万人死亡 国际吧最近，研究人员已经开始开发针对蛋白质-蛋白质相互作用的令人兴奋的药物 （PPI）调节转移驱动信号通路。PPI对于细胞信号转导至关重要， 在细胞的正常和异常功能中发挥关键作用。特异性和亲和力的变化， 相互作用可导致细胞功能障碍，例如不受控制的细胞生长和/或细胞迁移。一个关键 播散过程的关键是细胞协调信号传导途径与细胞骨架动力学的能力。 作为细胞骨架机制的关键协调者，GTP酶及其调节因子GEF是细胞骨架机制的关键参与者。 细胞运动许多肿瘤显示GTP酶和GEF的表达和/或活化增加。这些 蛋白质在癌症进展、转移和不良预后的几个方面起着重要作用。我们 长期目标是更好地了解GTP酶在转移过程中的分子机制， 设备更有效，毒性更小的乳腺癌疗法。我们实验室提供的强大的初步数据 揭示了一种新的蛋白质-蛋白质相互作用在细胞侵袭和转移中的关键作用。本申请的目的是 是实施试点高通量筛选（HTS），以确定这些新的结合的小分子抑制剂， 合作伙伴，将代表潜在的挽救生命的治疗线索，抑制癌症转移。三 本提案将制定具体目标：1）开发荧光测定法以检测该PPI; 2）试验 筛选：针对20，000种化合物的化合物集合进行完整的中试筛选，并进行后续测定; 3） 生物学效应：对活性化合物对细胞迁移和侵袭的影响进行初步表征。 我们的HTS将涉及一种创新的方法，分析细胞中的PPI并提高科学知识 细胞迁移中的关键信号蛋白。这项调查将具有重大意义， 理解细胞运动的基本和确定的特征，对生物学的许多领域有影响， 病理生理学

Abstract Breast cancer is a global problem, with 1.3 million new cases diagnosed and 430,000 deaths each year worldwide. Recently, researchers have begun developing exciting drugs to target the protein-protein interactions (PPIs) that regulate metastasis-driving signaling pathways. PPIs are critical for cellular signal transductions that play key roles in both normal and abnormal functions in cells. Changes in specificity and affinity of these interactions can lead to cellular malfunctions, such as uncontrolled cell growth and/or cell migration. A critical key in dissemination process is the ability of cells to coordinate signaling pathways with cytoskeleton dynamics. As critical coordinators of the cytoskeleton machinery, GTPases and their regulator GEFs are key players for cell movement. Many tumors show increased expression and/or activation of GTPases and GEFs. These proteins play fundamental roles in several aspects of cancer progression, metastasis and poor prognosis. Our long-term goals are to better understand the molecular mechanisms of GTPases in metastasis process and to device more effective, less toxic breast cancer therapies. Our strong preliminary data produced in our laboratory uncover a new protein-protein interaction critical in cell invasion and metastasis. The objective in this application is to implement a pilot high-throughput screen (HTS) to identify small molecule inhibitors of these novel binding partners that will represent potentially life-saving therapeutic leads for inhibiting cancer metastasis. Three specific aims will be developed in this proposal: 1) Development of fluorescence assay to detect this PPI; 2) Pilot screen: Complete pilot screen against a compound collection of 20,000 compounds and follow up assays; 3) Biological effects: perform initial characterization of active compounds on cell migration and invasion. Our HTS will involve an innovative approach that analyzes PPIs in cells and improve scientific knowledge about key signaling proteins in cell migration. This investigation will have substantial significance in understanding fundamental and defined features of cell motility, with implications for many areas of biology and pathophysiology.