Small Molecule Effectors of Eukaryotic Translation Initiation Site Selection

描述（申请人提供）：翻译起始是基因表达调控的关键环节。这个过程中的一个中心事件是mRNA中起始密码子的选择。如果翻译机制选择了错误的起始密码子，就会产生错误编码的蛋白质，这可能对细胞造成灾难性的后果。我的实验室已经开发了多种体外工具来研究蛋白质合成的关键步骤，并且与NIH/NICHD的Alan Hinnebusch实验室一起，我们最近在理解起始密码子识别的分子力学方面取得了重大进展。然而，我们缺乏的一套有价值的工具是可以通过翻译机制调节起始密码子识别保真度的小分子。为了满足这一需求，我们将使用双荧光素酶报告系统对酵母菌体内改变起始密码子识别保真度的化合物进行筛选。这些化合物中最有前途的将对其功效、毒性和一般作用方式进行评估。除了作为研究工具的价值外，在这个筛选中发现的化合物可能是药物开发的重要线索。许多严重遗传疾病的变异是由涉及起始密码子的突变引起的，这些疾病可能通过改变起始密码子识别的保真度的药物来改善，从而使具有异常起始位点的mRNA产生足够的蛋白质。改变起始密码子识别保真度的化合物也可能被证明对开发新的抗癌或抗真菌药物有用。这是一个高风险的项目，也是我实验室的一个新方向。然而，即使发现一种有效的化合物，也将是真核生物翻译研究领域的重大突破，并可能为未来的药物开发提供重要的起点。蛋白质是我们细胞的主力。它们完成维持细胞存活的基本任务，并允许细胞生长、分裂，并分化成特定类型的细胞，如心脏或肾脏细胞。蛋白质是由一种叫做核糖体的细胞机器制造的，核糖体读取我们基因中包含的信息来制造相应的蛋白质。一种叫做信使RNA （mRNA）的信使分子将基因的指令传递给核糖体。为了使指令有意义，核糖体需要在正确的起点开始读取mRNA。当细胞的生理变化或突变导致起点发生变化时，就会导致苯丙酮尿症、泰萨氏症和尿崩症等疾病。目前还没有已知的药物可以推动核糖体在改变的mrna上的正确点上启动，从而改善这些疾病和其他疾病。利用由美国国立卫生研究院和约翰霍普金斯大学医学院保存和维护的大量化合物，我们将寻找改变核糖体如何选择mRNA起始点的分子。这些化合物最终可能会被开发成药物，用于治疗诸如Tay-Sachs之类的疾病，以及某些类型的感染或癌症。这些化学物质也将是科学家们寻求更好地了解核糖体如何工作的宝贵工具。对这种复杂的细胞机器的更深入的了解可能会导致新的方法来治疗影响核糖体功能的无数疾病。

DESCRIPTION (provided by applicant): Translation initiation is a key point in the regulation of gene expression. A central event in this process is the selection of the initiation codon in the mRNA. If the incorrect initiation codon is selected by the translational machinery, a miscoded protein will be produced, which could have disastrous consequences for the cell. My lab has developed a variety of in vitro tools for studying this key step in protein synthesis, and, together with Alan Hinnebusch's lab at the NIH/NICHD, we have recently made significant strides in understanding the molecular mechanics of start codon recognition. One valuable set of tools we lack, however, are small molecules that can modulate the fidelity of initiation codon recognition by the translational machinery. To address this need, we will conduct a screen for compounds that alter the fidelity of initiation codon recognition in vivo in yeast using a dual luciferase reporter system. The most promising of these compounds will be evaluated for efficacy, toxicity and general mode of action. In addition to their value as research tools, compounds discovered in this screen would be potentially important leads for drug development. Variants of many serious genetic diseases are caused by mutations involving initiation codons, and it is possible that these diseases could be ameliorated by drugs that alter the fidelity of start codon recognition, allowing sufficient protein to be produced from the mRNA with the aberrant start site. Compounds that alter the fidelity of start codon recognition might also prove useful for the development of novel anticancer or antifungal agents. This is a high-risk project and a new direction for my lab. However, if even one effective compound is discovered it would be a major breakthrough for the field of eukaryotic translation research and could provide an important starting point for future drug development. Proteins are the workhorses of our cells. They do the essential tasks that keep cells alive and allow them to grow, divide, and specialize into specific types, such as heart or kidney cells. Proteins are made by a cellular machine called the ribosome that reads information contained in our genes to make the corresponding proteins. A messenger molecule called messenger RNA (mRNA) carries a gene's instructions to the ribosome. For the instructions to make sense, the ribosome needs to start reading the mRNA at the correct starting point. When physiological changes in the cell or mutations cause a change in the starting point, diseases such as phenylketonuria, Tay-Sachs and diabetes insipidus can result. Currently there are no known drugs that can push the ribosome to start at the correct point on altered mRNAs, ameliorating these and other diseases. Using a vast collection of chemical compounds housed and maintained by the National Institutes of Health and Johns Hopkins University School of Medicine, we will search for molecules that alter how the ribosome selects the starting point in the mRNA. These compounds could ultimately be developed into drugs to treat diseases such as Tay-Sachs, as well as certain types of infections or cancer. These chemicals would also be valuable tools for scientists seeking to better understand how the ribosome works. A deeper understanding of this complex cellular machine could lead to new approaches to treat the myriad diseases that affect the ribosome's function.