High Throughput Assays Targeting Ribosome Recruitment

针对核糖体招募的高通量检测

基本信息

批准号：
7212152
负责人：
JERRY PELLETIER
金额：
$ 16.18万
依托单位：
MCGILL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-04-01 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7212152
关键词：
1-Phosphatidylinositol 3-Kinase 5&apos Untranslated Regions Amino Acids Apoptotic Applications Grants Arginine Binding Biological Assay Biological Process Biology C-terminal Cancer Model Cell Cycle Progression Cell physiology Cells Chemicals Class Clinical Trials Complex Cues Discrimination Dissociation Figs - dietary Fluorescence Fluorescence Polarization Fluorescence Resonance Energy Transfer Foundations Human Hypoxia Insulin Lead Malignant - descriptor Malignant Neoplasms Messenger RNA Molecular Monitor Mus N-terminal Neoplastic Cell Transformation Nucleotides Pathway interactions Peptide Initiation Factors Play Process Property Protein Biosynthesis Proteins RNA Binding RNA Cap-Binding Proteins RNA Helicase RNA Recognition Motif RNA-Binding Proteins Rate Recruitment Activity Regulation Ribosomes Role Series Signal Pathway Sirolimus Starvation Structure Testing Translation Initiation Translation Process Translations base cell growth regulation design helicase high throughput screening human FRAP1 protein in vivo inhibitor/antagonist insight mRNA capping prevent programs response small molecule translation factor tumor progression tumorigenesis

1-Phosphatidylinositol 3-Kinase 5&apos Untranslated Regions Amino Acids Apoptotic Applications Grants Arginine Binding Biological Assay Biological Process Biology C-terminal Cancer Model Cell Cycle Progression Cell physiology Cells Chemicals Class Clinical Trials Complex Cues Discrimination Dissociation Figs - dietary Fluorescence Fluorescence Polarization Fluorescence Resonance Energy Transfer Foundations Human Hypoxia Insulin Lead Malignant - descriptor Malignant Neoplasms Messenger RNA Molecular Monitor Mus N-terminal Neoplastic Cell Transformation Nucleotides Pathway interactions Peptide Initiation Factors Play Process Property Protein Biosynthesis Proteins RNA Binding RNA Cap-Binding Proteins RNA Helicase RNA Recognition Motif RNA-Binding Proteins Rate Recruitment Activity Regulation Ribosomes Role Series Signal Pathway Sirolimus Starvation Structure Testing Translation Initiation Translation Process Translations base cell growth regulation design helicase high throughput screening human FRAP1 protein in vivo inhibitor/antagonist insight mRNA capping prevent programs response small molecule translation factor tumor progression tumorigenesis

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项目摘要

DESCRIPTION (provided by applicant): Translation is an essential cellular process whose deregulation is associated with alterations in cell growth regulation, cell cycle progression, and apoptotic responses. There is much evidence supporting the notion that aberrant control of protein translation contributes to neoplastic transformation. Signaling pathways (e.g. - ras and Akt) that regulate the translational machinery are activated in many human cells, over-expression of certain translation factors can lead to malignant transformation, and several components of the translational apparatus are over-expressed in human cancers. Indeed, elevated expression levels of elF4E (the mRNA cap binding protein involved in ribosome recruitment) leads to transformation in murine cancer model and is implicated in chemoresistance. Rapamycin, an inhibitor of the ribosome recruitment step, shows significant anti-cancer activity and is currently being tested in clinical trials. In this grant application, we propose to develop a series of HTS assays that will form the foundation of a chemical biology program aimed at better understanding the mechanism of translation, as well as the role that deregulation of this process plays in tumor progression. The HTS assays will target translation initiation factors involved in eukaryotic ribosome recruitment and will be used to identify compounds that can specifically inhibit this process. Our specific aims are to: i) target the interaction between elF4E and the mRNA cap structure for HTS assay design; ii) develop an HTS assay that monitors the helicase activity of elF4A, an ATP-dependent RNA helicase that unwinds mRNA secondary structure in the 5' UTR of mRNAs; iii) develop an HTS assay for inhibitors of elF4B activity, an RNA binding factor that functions in conjunction with elF4A to facilitate mRNA/ribosome binding; and iv) develop an HTS assay to prevent formation of the elF4E/4E-BP inhibitory complex, a heterodimer whose formation is stabilized by rapamycin.

描述（由申请人提供）：翻译是一个必不可少的细胞过程，其放松管制与细胞生长调节，细胞周期进程和凋亡反应的改变有关。有很多证据支持这样一个观念，即对蛋白质翻译的异常控制有助于肿瘤转化。在许多人类细胞中激活了调节翻译机制的信号传导途径（例如-RAS和AKT），某些翻译因子的过表达可能导致恶性转化，而翻译设备的几个成分在人类癌症中被过表达。实际上，ELF4E的表达水平升高（参与核糖体募集的mRNA帽结合蛋白）导致鼠类癌模型的转化，并与化学抗性有关。雷帕霉素是核糖体募集步骤的抑制剂，它显示出明显的抗癌活性，目前正在临床试验中进行测试。在此赠款应用中，我们建议开发一系列的HTS测定法，这些测定法将构成旨在更好地理解翻译机理的化学生物学计划的基础，以及该过程在肿瘤进展中起作用的作用。 HTS分析将针对真核核糖体募集中涉及的翻译起始因子，并将用于识别可以特异性抑制此过程的化合物。我们的具体目的是：i）针对HTS分析设计的ELF4E与mRNA CAP结构之间的相互作用； ii）开发了HTS测定法，该测定法对ATP依赖性RNA解旋酶的解旋酶活性进行了监测，该酶在mRNA的5'UTR中放弃mRNA二级结构； iii）开发用于ELF4B活性抑制剂的HTS分析，这是一种与ELF4A结合起作用以促进mRNA/核糖体结合的RNA结合因子；和iv）开发HTS测定法，以防止形成ELF4E/4E-BP抑制复合物，这是一种杂质二聚体，其形成被雷帕霉素稳定。