Development of High Throughput Screening Assays (RMI)

高通量筛选分析 (RMI) 的开发

• 批准号: 7162817
• 负责人: STEVEN S ROSENFELD
• 金额: $ 6.66万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2007-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7162817
• 关键词: adenosine triphosphate; cell cycle; cell motility; chemical registry /resource; drug administration rate /duration; drug discovery /isolation; drug screening /evaluation; enzyme inhibitors; fluorescence resonance energy transfer; glioma; high throughput technology; hydrolysis; intracellular transport; kinesin; myosins; neoplasm /cancer pharmacology; neuropharmacology; pharmacokinetics; small molecule; technology /technique development

DESCRIPTION (provided by applicant): The prognosis for patients afflicted with malignant gliomas remains grim in spite of many years of intensive clinical research. Several factors contribute to this state of affairs. First, these tumors are highly invasive. This invasiveness greatly limits the effectiveness of local therapies, such as surgery, radiosurgery, and brachytherapy. Second, these tumors are very effective at generating their own blood supply. They accomplish this by secreting a variety of factors that induce endothelial cells to migrate toward and invade into tumor stroma. Finally, gliomas, like other malignancies, produce a variety of growth factors that in aggregate stimulate their mitotic activity--one that is relatively resistant to cell cycle-specific chemotherapies. While it may not seem immediately obvious, tumor invasion, angiogenesis, and mitosis have one thing in common--all depend critically on cell motility. Cell motility allows glioma and endothelial cells to crawl through the extracellular matrix of the brain. However, cell motility also includes movement of intracellular components from one location within the cell to another, such as occurs in mitosis, where chromosomes are actively translocated to the poles. All of these motile processes depend upon a class of enzymes, called "molecular motors", which utilize the energy of ATP hydrolysis to generate movement. While it would seem very reasonable to treat gliomas by targeting their myosins and kinesins, the pharmacologic inhibition of molecular motors as a form of cancer therapy has been largely unexplored and unexploited. I will address this issue by proposing to identify new inhibitors of myosin II and kinesin by developing high throughput screening assays based on a series of novel spectroscopic probes developed in my laboratory. These assays will be performed at the High Throughput Screening (HTS) Laboratory at Southern Research, Inc. and will be used to identify promising compounds identified from screening several large chemical libraries (Aim 1). Compounds identified in Aim 1 will be validated by fluorescence spectroscopy and transient state kinetics to determine the mechanism of inhibition and its specificity (Aim 2). This work will serve as the basis for a future extramural funding initiative designed to take promising compounds identified by this approach into pre-clinical glioma model systems, and ultimately, into clinical trial.

描述（由申请人提供）：尽管经过多年深入的临床研究，患有恶性神经胶质瘤的患者的预后仍然严峻。 有几个因素导致了这种状况。 首先，这些肿瘤具有高度侵袭性。 这种侵入性极大地限制了局部治疗的有效性，例如手术、放射外科和近距离放射治疗。 其次，这些肿瘤能够非常有效地产生自己的血液供应。 它们通过分泌多种诱导内皮细胞迁移并侵入肿瘤基质的因子来实现这一点。 最后，与其他恶性肿瘤一样，神经胶质瘤会产生多种生长因子，这些生长因子总体上会刺激其有丝分裂活性，而这种活性对细胞周期特异性化疗相对具有抵抗力。 虽然看起来可能不是很明显，但肿瘤侵袭、血管生成和有丝分裂有一个共同点——所有这些都严重依赖于细胞运动。 细胞运动使神经胶质瘤和内皮细胞爬过大脑的细胞外基质。 然而，细胞运动还包括细胞内成分从细胞内的一个位置移动到另一个位置，例如发生在有丝分裂中，其中染色体主动易位到两极。 所有这些运动过程都依赖于一类称为“分子马达”的酶，它们利用 ATP 水解的能量来产生运动。 虽然通过靶向肌球蛋白和驱动蛋白来治疗神经胶质瘤似乎非常合理，但分子马达的药理学抑制作为癌症治疗的一种形式在很大程度上尚未被探索和利用。 我将提出通过基于我实验室开发的一系列新型光谱探针开发高通量筛选测定来鉴定新的肌球蛋白 II 和驱动蛋白抑制剂来解决这个问题。 这些测定将在 Southern Research, Inc. 的高通量筛选 (HTS) 实验室进行，并将用于鉴定通过筛选几个大型化学库（目标 1）而确定的有前景的化合物。 目标 1 中鉴定的化合物将通过荧光光谱和瞬态动力学进行验证，以确定抑制机制及其特异性（目标 2）。 这项工作将作为未来外部资助计划的基础，旨在将通过这种方法鉴定的有前景的化合物纳入临床前神经胶质瘤模型系统，并最终进入临床试验。