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

开发基于细胞的高含量 mTOR 信号抑制剂筛选

• 批准号: 7290648
• 负责人: JOHN BLENIS
• 金额: $ 21.15万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-05 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7290648
• 关键词: Address; Amino Acids; Antifungal Agents; Antineoplastic Agents; Benign; Biochemical; Biological Assay; Biological Factors; Biology; CCI-779; Cell physiology; Cells; Cellular Morphology; Chemicals; Chemistry; Classification; Clinical Trials; Collection; Data; Defect; Detection; Development; Disease; Disease Progression; Eligibility Determination; Event; Feedback; Goals; Growth; Growth Factor; Image; Immunosuppressive Agents; Institutes; Lead; Libraries; Macrolide Antibiotics; Malignant Neoplasms; Measures; Mediating; Metabolic Diseases; Microscopy; Molecular; Mutation; Numbers; Nutrient; Pathway interactions; Pharmaceutical Preparations; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Pilot Projects; Proteins; Purpose; Regulation; Reproducibility; Research; Ribosomal Protein S6; Screening procedure; Series; Signal Transduction; Sirolimus; Syndrome; Techniques; Therapeutic; Work; base; cancer cell; cancer therapy; cell growth; clinical application; high throughput screening; human disease; improved; inhibitor/antagonist; interest; kinase inhibitor; novel; response; small molecule; therapeutic target; tissue/cell culture; tool; tumor; tumor progression; wortmannin

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.

描述（由申请人提供）：哺乳动物雷帕霉素靶标（mTOR）是细胞生长和增殖的关键调节因子，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。组成型、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，雷帕霉素衍生物抑制 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 信号传导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 的反馈环路，表明在治疗与 mTOR 信号传导网络失调相关的疾病中迫切需要额外的治疗途径。为了促进识别其他靶向 mTOR 的小分子，我们将开发一种高通量、基于细胞的测定法，用于定量检测 rpS6 磷酸化，作为 mTOR 通路激活状态的衡量标准。使用该测定，我们将进行一系列试点研究，从已知的 mTOR 信号传导抑制剂（例如雷帕霉素和渥曼青霉素）开始，最终对包含 200 种市售激酶抑制剂的库以及更大的化学多样化生物活性化合物库进行试点筛选。此外，我们将开发一系列二次测定，结合高内涵成像、生化测定和基于细胞的功能读数，以评估我们的高通量测定中确定的靶标调节 mTOR 信号传导的能力。我们将特别强调鉴定能够作为“广谱”mTOR 抑制剂阻断 mTORC1 和 mTORC2 活性的化合物。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，还可以促进涉及 mTOR 信号传导过度活跃的疾病的治疗。在许多癌症中，细胞中一种称为 mTOR 的关键蛋白的调节缺陷直接导致恶性细胞生长失控。雷帕霉素是一种药物，可阻断 mTOR 的部分（但不是全部）细胞功能，并在癌症治疗中显示出一些前景，但并不总是有效。我们的工作旨在寻找新的药物来特异性抑制 mTOR 活性的各个方面，目标是改善涉及 mTOR 信号传导错误调节的癌症的治疗。