Development of High Throughput Screening Assays (RMI)

描述（由申请人提供）：尽管经过多年的深入临床研究，恶性胶质瘤患者的预后仍然严峻。 造成这种状况的因素有几个。 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，例如外科手术、放射外科手术和近距离放射治疗。 其次，这些肿瘤在产生自身血液供应方面非常有效。 它们通过分泌诱导内皮细胞向肿瘤间质迁移和侵入肿瘤间质的各种因子来实现这一点。 最后，神经胶质瘤，像其他恶性肿瘤一样，产生多种生长因子，这些生长因子共同刺激它们的有丝分裂活性--一种对细胞周期特异性化疗相对具有抗性的生长因子。 虽然这似乎并不明显，但肿瘤侵袭、血管生成和有丝分裂有一个共同点--都严重依赖于细胞运动。 细胞运动性允许胶质瘤和内皮细胞爬行通过脑的细胞外基质。 然而，细胞运动性还包括细胞内组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胶质瘤似乎是非常合理的，但作为癌症治疗形式的分子马达的药理学抑制在很大程度上尚未探索和利用。 我将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识别肌球蛋白II和驱动蛋白的新抑制剂来解决这个问题。 这些试验将在Southern Research，Inc.的高通量筛选（HTS）实验室进行。并将用于鉴定从筛选几个大型化学文库中鉴定的有前景的化合物（Aim 1）。 将通过荧光光谱和瞬态动力学验证目标1中鉴别的化合物，以确定抑制机制及其特异性（目标2）。 这项工作将作为未来校外资助计划的基础，旨在将通过这种方法鉴定的有前途的化合物纳入临床前胶质瘤模型系统，并最终进入临床试验。

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.