Development of a high content cell based screen for inhibitors of the mTOR signal

描述（由申请人提供）：哺乳动物雷帕霉素靶点（mTOR）是细胞生长和增殖的关键调节剂，是多种稳态输入的中心整合点，包括生长因子可用性、能量水平和氨基酸充足性。组成性的、不受调节的mTOR激酶活性几乎是癌细胞的普遍特征，mTOR信号网络的缺陷也与代谢紊乱和良性肿瘤综合征有关。由于这些原因，人们对天然产物雷帕霉素衍生物的临床应用产生了浓厚的兴趣，雷帕霉素可以抑制mtor介导的信号转导的某些方面。虽然雷帕霉素在临床试验中显示出作为抗癌药物的前景，但mTOR信号的雷帕霉素不敏感方面对癌症进展的潜在贡献，包括最近发现的涉及mTOR直接激活Akt的反馈回路，表明迫切需要额外的治疗途径来治疗与mTOR信号网络失调相关的疾病。为了便于鉴定靶向mTOR的其他小分子，我们将开发一种高通量、基于细胞的定量检测rpS6磷酸化的方法，作为mTOR途径激活状态的测量。使用这种方法，我们将进行一系列的试点研究，从已知的mTOR信号抑制剂开始，如雷帕霉素和wortmannin，最终在包含200个市售激酶抑制剂的文库中进行试点筛选，以及更大的化学多样性生物活性化合物文库。此外，我们将开发一系列二级分析，结合高含量成像、生化分析和基于细胞的功能读数，以评估在我们的高通量分析中确定的靶标调节mTOR信号的能力。重点将放在鉴定能够作为“广谱”mTOR抑制剂的化合物上，这些抑制剂可以阻断mTORC1和mTORC2的活性。通过这项工作确定的特异性抑制剂不仅可以作为有价值的研究工具，而且可以推进涉及mTOR信号过度活跃的疾病的治疗。在许多癌症中，细胞中一种被称为mTOR的关键蛋白的调控缺陷直接导致了恶性细胞不受控制的生长。雷帕霉素是一种阻断mTOR部分（但不是全部）细胞功能的药物，在癌症治疗中显示出一些希望，但并不总是有效。我们的工作旨在寻找特异性抑制mTOR活性各方面的新药，以改善涉及mTOR信号失调的癌症的治疗方法。

DESCRIPTION (provided by applicant): Mammalian target of rapamycin (mTOR) is a critical regulator of cell growth and proliferation, serving as the central integration point for multiple homeostatic inputs, including growth factor availability, energy levels and amino-acid sufficiency. Constitutive, unregulated mTOR kinase activity is a nearly universal feature of cancer cells, and defects in the mTOR signaling network have also been implicated in metabolic disorders and benign tumor syndromes. For these reasons there has been intense interest in the clinical application of derivatives of the natural product rapamycin which inhibits some aspects of mTOR-mediated signal transduction. While rapamycin has shown promise as an anti-cancer agent in clinical trials, the potential contribution of rapamycin-insensitive aspects of mTOR signaling to cancer progression, including the recently identified feedback loop involving the direct activation of Akt by mTOR, points to the critical need for additional therapeutic avenues in the treatment of diseases associated with deregulation of the mTOR signaling network. In order facilitate the identification of additional small molecules targeting mTOR, we will develop a high throughput, cell-based assay for the quantitative detection of rpS6 phosphorylation as a measure of the activation state of the mTOR pathway. Using this assay, we will perform a series of pilot studies, starting with known inhibitors of mTOR signaling, such as rapamycin and wortmannin, and culminating in pilot screens of a library comprising 200 commercially available kinase inhibitors, as well as a larger library of chemically diverse bioactive compounds. In addition, we will develop a series of secondary assays, combining high content imaging, biochemical assays, and cell based functional readouts, to evaluate targets identified in our high throughput assay for their ability to modulate mTOR signaling. Particular emphasis will be placed on identifying compounds that are able to act as 'broad spectrum' mTOR inhibitors that block the activity of both mTORC1 and mTORC2. Specific inhibitors identified through this work will not only serve as valuable research tools but may advance the treatment of diseases involving hyperactive mTOR signaling. In many cancers, the defective regulation of a key protein in the cell known as mTOR directly contributes to the uncontrolled growth of malignant cells. Rapamycin is a drug that blocks some, but not all, cellular functions of mTOR and has shown some promise in cancer treatment, but it is not always effective. Our work is aimed at finding new drugs to specifically inhibit all aspects of mTOR activity, with the goal of improving treatments for cancers involving mis-regulation of mTOR signaling.