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

描述(申请人提供)：基因被转录以产生信使RNA(信使RNA)分子，然后被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，以此作为最终在这一领域发现新的治疗选择的手段。我们关注的是聚(A)结合蛋白(PABP)，它与信使核糖核酸的尾部结合，在蛋白质翻译和信使核糖核酸降解中起核心作用。PABP是细胞蛋白质生产所必需的，是一个主要的控制位点，通过与其他调节和信号蛋白的结合相互作用而发挥作用。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，这两个口袋也是潜在的药物结合部位。我们的研究计划是与美国国立卫生研究院合作筛选大型化学库，以发现将结合到这些口袋中的类药物化合物。我们的筛查是PABP的调节域与一个小蛋白片段之间的竞争分析，该小蛋白片段来自一个关键的相互作用伙伴，并被荧光标记。由于最初的筛选结果往往是假阳性，我们将用对照试验筛选出非特异性的结果和不溶性化合物，并使用可以测量蛋白质-药物相互作用的灵敏检测方法来确认PABP的直接结合。验证的HIT化合物的结合位点将使用高分辨率核磁共振成像绘制到PABP的表面，以查看化合物如何相互作用，并检查它们干扰PABP调控相互作用的方式。如果我们确定具有良好效力的类药物化合物，我们将在细胞提取物和整个细胞中测试它们，看看它们如何改变翻译和信使核糖核酸的降解过程。我们还将直接研究PABP相互作用对化合物添加的响应变化。我们的假设是，我们将把小的化学探针作为有用的工具，来解开翻译和mRNA降解的复杂过程。对细胞力学的进一步了解将有助于洞察哪些PABP相互作用将是与蛋白质产生过多或过少相关疾病的最佳治疗靶点。 与公共健康相关：DNA基因被转录成信使RNA分子，然后被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聚(A)结合蛋白的潜在治疗靶点，它在翻译和信使RNA稳定性方面发挥着重要作用。我们打算筛选调节这种蛋白质的化合物，以产生一种探针，有助于研究这一关键的生物学过程，并有望及时为患者带来新药。

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.