HTS Screening for Small-Molecule Inhibitors of miRNA-Mediated mRNA Deadenylation

HTS 筛选 miRNA 介导的 mRNA 脱腺苷酸化的小分子抑制剂

• 批准号: 8251893
• 负责人: Kalle B Gehring
• 金额: $ 0.14万
• 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/8251893
• 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; public health relevance; 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）：基因转录产生信使RNA（mRNA）分子，然后翻译成蛋白质的过程对细胞的生命和生长至关重要。这些关键的细胞功能在癌症和其他疾病中经常改变，我们有兴趣增加我们对这些过程的了解，作为最终发现该领域新治疗方案的手段。我们专注于多聚腺苷酸结合蛋白（PABP），它结合到mRNA的尾巴，并在蛋白质翻译和mRNA降解中发挥核心作用。PABP是细胞蛋白质生产所必需的，并且是通过与其他调节和信号蛋白的结合相互作用发挥功能的主要控制位点。我们已经制作了这些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，也是潜在的药物结合位点。我们的研究计划是与NIH合作筛选大型化学库，以发现与这些口袋结合的药物样化合物。我们的筛选是PABP的调控结构域和一个小的蛋白质片段之间的竞争测定，该蛋白质片段来自荧光标记的关键相互作用伴侣。由于最初的筛选命中经常是假阳性，我们将过滤出非特异性命中和不溶性化合物与控制测定，并确认直接PABP结合使用灵敏的检测方法，可以测量蛋白质-药物相互作用。使用高分辨率NMR成像将经验证的命中化合物的结合位点映射到PABP表面上，以观察化合物如何相互作用，并检查它们干扰PABP调节相互作用的方式。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全细胞中测试它们，以观察它们如何改变翻译和mRNA降解的过程。我们还将直接研究PABP相互作用的变化，以响应化合物添加。我们的假设是，小的化学探针，我们将作为有用的工具，在解开翻译和mRNA降解的复杂过程。对细胞力学的进一步了解将有助于了解PABP相互作用在与蛋白质产生过多或过少相关的疾病中是最佳治疗靶点。 公共卫生相关性：DNA基因转录产生信使RNA分子，然后翻译成蛋白质的过程对细胞的生命和生长至关重要。这些功能在癌症和其他疾病中经常发生变化，我们有兴趣研究一种名为poly（A）结合蛋白的潜在治疗靶点，它在翻译和信使RNA稳定性中起着重要作用。我们打算筛选能够调节这种蛋白质的化合物，以产生一种探针，帮助研究这一关键的生物学过程，并希望及时为患者带来新药。