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

基本信息

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
项目类别：
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

项目摘要

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）：雷帕霉素的哺乳动物靶标（MTOR）是细胞生长和增殖的关键调节剂，它是多种稳态输入的中心整合点，包括生长因子的可用性，能量水平和氨基酸足够。组成型，不调节的MTOR激酶活性是癌细胞的几乎普遍特征，MTOR信号网络中的缺陷也与代谢性疾病和良性肿瘤综合征有关。由于这些原因，对天然产物雷帕霉素的衍生物的临床应用引起了极大的兴趣，雷帕霉素抑制了mTOR介导的信号转导的某些方面。雷帕霉素在临床试验中表现出作为抗癌剂的希望，但MTOR信号传导不敏感的方面对癌症进展的潜在贡献，包括最近确定的反馈回路，涉及MTOR直接激活AKT，这表明了与MTESESESENESESESENESESESESESENESESENESESESESENESENESESESERESESS IDERESESENESERESENESSENERESENESSENEREDERESESS IDEREDERESENERSENESENESENESENESENESENESESESENS互动相关的反馈循环。为了促进靶向MTOR的其他小分子，我们将开发出一种基于细胞的吞吐量，用于定量检测RPS6磷酸化，以作为MTOR途径激活状态的度量。使用此测定，我们将从已知的MTOR信号传导抑制剂（例如雷帕霉素和磨牙）进行一系列试验研究，并在包含200个商业可用激酶抑制剂的图书馆的试验筛选中达到顶点，以及更大的化学化学复合化合物库。此外，我们将开发一系列辅助测定法，结合高含量成像，生化测定和基于细胞的功能读数，以评估我们在高吞吐量测定中确定的目标，以调节其调节MTOR信号传导的能力。特别重点将放在识别能够充当“广泛” MTOR抑制剂的化合物上，从而阻止MTORC1和MTORC2的活性。通过这项工作确定的特定抑制剂不仅可以用作有价值的研究工具，而且可能会进步涉及多动MTOR信号传导的疾病。在许多癌症中，被称为MTOR的细胞中关键蛋白的有缺陷调节直接导致恶性细胞的不受控制的生长。雷帕霉素是一种阻止MTOR的一些但不是全部细胞功能的药物，并且在癌症治疗方面表现出了一些希望，但并不总是有效的。我们的工作旨在寻找新药物以特别抑制MTOR活性的各个方面，目的是改善涉及MTOR信号不良调节的癌症的治疗方法。