HTS Screening for small-molecule inhibitors of microRNA-mediated mRNA deadenylati

HTS 筛选 microRNA 介导的 mRNA 去腺苷酸小分子抑制剂

• 批准号: 8139546
• 负责人: Kalle B Gehring
• 金额: $ 1.88万
• 依托单位: MCGILL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-07 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8139546
• 关键词: Affect; Affinity; Area; Binding; Binding Sites; Biological Assay; Biological Process; C-terminal; Cell physiology; Cells; Chemicals; Complex; Consensus Sequence; DNA; Data; Detection; Disease; Drug Binding Site; Drug Interactions; Ensure; Fluorescence; Fluorescence Polarization; Genes; Goals; Image; In Vitro; Inhibitory Concentration 50; Knowledge; Label; Lead; Libraries; Life; Magnetic Resonance Imaging; Malignant Neoplasms; Maps; Measures; Mechanics; Mediating; Messenger RNA; Methods; MicroRNAs; NMR Spectroscopy; Patients; Peptides; Pharmaceutical Preparations; Play; Poly(A) Tail; Poly(A)-Binding Proteins; Process; Production; Protein Binding; Protein C; Protein Fragment; Proteins; RNA Degradation; RNA Interference; RNA Stability; Reading; Regulation; Reproducibility; Research; Resolution; Role; SHPS-1 protein; Screening procedure; Site; Specificity; Surface; Tail; Testing; Time; Titrations; Translating; Translation Initiation; Translation Process; Translations; United States National Institutes of Health; X-Ray Crystallography; base; cell growth; improved; in vivo; inhibitor/antagonist; insight; interest; light scattering; nephelometry; novel therapeutics; response; small molecule; small molecule libraries; stoichiometry; therapeutic target; tool

DESCRIPTION (provided by applicant): The processes by which genes are transcribed to produce messenger RNA (mRNA) molecules, and then translated into proteins are central to the life and growth of cells. These key cellular functions are frequently altered in cancers and other diseases, and we are interested in increasing our knowledge of these processes as a means of ultimately discovering new therapeutic options in this area. We are focused on the poly (A)-binding protein (PABP) which binds to the mRNA tail and plays a central role in protein translation and mRNA degradation. PABP is essential for cellular protein production and is a major control site that functions via binding interactions with other regulatory and signaling proteins. We have produced high-resolution images of these PABP interactions and have identified two critical binding pockets that are also potential drug binding sites. Our research plan is to screen large chemical libraries in cooperation with the NIH to discover drug-like compounds that will bind to these pockets. Our screen is a competition assay between the regulatory domain of PABP and a small protein fragment, derived from a key interacting partner that is fluorescently labeled. Since initial screening hits are frequently false positives, we will filter out non-specific hits and insoluble compounds with control assays, and confirm direct PABP binding using sensitive detection methods that can measure protein-drug interactions. The binding sites of validated hit compounds will be mapped onto the surface of PABP using high resolution NMR imaging to see how the chemical compounds interact, and examine the means by which they interfere with PABP regulatory interactions. If we identify drug-like compound with good potency we will test them in cellular extracts and whole cells to see how they alter the processes of translation and mRNA degradation. We will also directly examine changes in PABP interactions in response to compound addition. Our hypothesis is that small chemical probes we will serve as useful tools in unraveling the complex processes of translation and mRNA degradation. An increased understanding of cellular mechanics will provide insight into which PABP interactions would be the best therapeutic targets in diseases associated with too much or too little protein production. PUBLIC HEALTH RELEVANCE: The processes by which DNA genes are transcribed to produce messenger RNA molecules, and then translated into proteins is central to the life and growth of cells. These functions are frequently altered in cancer and other diseases and we are interested in examining a potential therapeutic target called the poly (A)-binding protein, which plays a major role in translation and messenger RNA stability. We intend to screen for chemical compounds that will modulate this protein to produce a probe that will help investigate this key biological process, and hopefully in time lead to new drugs for patients.

描述（由申请人提供）：转录基因产生信使RNA（mRNA）分子的过程，然后转化为蛋白质是细胞生命和生长的核心。这些关键的细胞功能在癌症和其他疾病中经常发生变化，我们有兴趣增加对这些过程的了解，以此作为最终在该领域发现新的治疗选择的手段。我们专注于与mRNA尾巴结合并在蛋白质翻译和mRNA降解中起核心作用的聚（A）结合蛋白（PABP）。 PABP对于细胞蛋白的产生至关重要，是一个主要控制位点，通过与其他调节和信号蛋白的结合起作用。我们已经产生了这些PABP相互作用的高分辨率图像，并确定了两个关键的结合口袋，它们也是潜在的药物结合位点。我们的研究计划是与NIH合作筛选大型化学文库，以发现将与这些口袋结合的药物样化合物。我们的屏幕是PABP调节域和小蛋白质片段之间的竞争测定法，该蛋白质片段源自荧光标记的关键相互作用伴侣。由于最初的筛选命中率通常是假阳性，因此我们将使用控制测定的非特异性命中和不溶性化合物，并使用敏感检测方法确认直接的PABP结合，该方法可以测量蛋白质 - 药水相互作用。经过验证的HIT化合物的结合位点将使用高分辨率NMR成像映射到PABP的表面上，以查看化学化合物如何相互作用，并检查它们干扰PABP调节相互作用的手段。如果我们鉴定出具有良好效力的药物样化合物，我们将在细胞提取物和整个细胞中测试它们，以了解它们如何改变翻译和mRNA降解过程。我们还将直接检查PABP相互作用的变化，以响应化合物添加。我们的假设是，小型化学探针我们将用作阐明复杂翻译和mRNA降解过程的有用工具。对细胞力学的越来越多的理解将提供有关哪些PABP相互作用将是与蛋白质产生过多或太少有关的疾病中最好的治疗靶标。 公共卫生相关性：DNA基因转录以产生信使RNA分子的过程，然后转化为蛋白质是细胞生命和生长的核心。这些功能在癌症和其他疾病中经常发生改变，我们有兴趣检查一个称为poly（a）结合蛋白的潜在治疗靶标，该蛋白在翻译和使者RNA稳定性中起着重要作用。我们打算筛选化合物，这些化合物将调节该蛋白质以产生有助于研究这一关键生物学过程的探针，并希望及时为患者提供新药。