Small Molecule Effectors of Eukaryotic Translation Initiation Site Selection

描述(申请人提供)：翻译启动是基因表达调控的一个关键点。这一过程中的一个中心事件是选择mRNA中的起始密码子。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，这可能会给细胞带来灾难性的后果。我的实验室已经开发了各种体外工具来研究蛋白质合成中的这一关键步骤，并与NIH/NICHD的Alan Hinnebusch实验室一起，最近在理解起始密码子识别的分子机制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小分子，它们可以通过翻译机制调节起始密码子识别的保真度。为了满足这一需求，我们将使用双重荧光素酶报告系统在酵母中筛选能够改变体内起始密码子识别保真度的化合物。将对这些化合物中最有希望的化合物进行有效性、毒性和一般作用模式的评估。除了它们作为研究工具的价值外，在这一筛选中发现的化合物可能是药物开发的重要线索。许多严重遗传性疾病的变异是由涉及起始密码子的突变引起的，这些疾病有可能通过改变起始密码子识别的保真度的药物来改善，允许从具有异常起始密码子的信使核糖核酸产生足够的蛋白质。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型抗癌或抗真菌药物有用。这是一个高风险的项目，也是我的实验室的一个新方向。然而，如果发现一种有效的化合物，这将是真核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，并允许它们生长、分裂并专门化成特定类型，如心脏或肾脏细胞。蛋白质是由一种名为核糖体的细胞机器制造的，它读取我们基因中包含的信息来制造相应的蛋白质。一种名为信使RNA(信使RNA)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，核糖体需要从正确的起始点开始读取mRNA。当细胞的生理变化或突变引起起始点的变化时，可能会导致苯丙酮尿症、泰萨氏病和尿崩症等疾病。目前还没有已知的药物可以推动核糖体从改变的mRNAs的正确位置开始，从而改善这些和其他疾病。利用美国国立卫生研究院和约翰霍普金斯大学医学院收藏和维护的大量化合物，我们将寻找改变核糖体如何选择信使核糖核酸起始点的分子。这些化合物最终可能被开发成治疗泰-萨克斯等疾病以及某些类型的感染或癌症的药物。对于寻求更好地了解核糖体如何工作的科学家来说，这些化学物质也将是有价值的工具。对这种复杂的细胞机器的深入了解可能会导致治疗影响核糖体功能的无数疾病的新方法。

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.