Modulators of the fidelity of start codon recognition in eukaryotes

描述(申请人提供)：真核翻译机制对起始密码子的识别是基因表达的关键步骤。它设置了mRNA将在其中被解码的阅读框架，并定义了相应蛋白质的N末端。我们开发了一种体内高通量双荧光素酶检测方法，可以准确测量酿酒酵母起始密码子识别的保真度。使用这一分析和一种强大的二次分析来消除通常影响一种或两种荧光素酶的表达或活性的试剂，我们筛选了50,000多个分子，以寻找降低起始密码子识别保真度的化合物。从这个筛选中发现了两个结构相似的分子，它们增加了对近源密码子的使用(例如，UUG、CUG等)。作为起始点。这些活性在几个三级分析中得到了证实。对这些化合物的体内效应的进一步分析支持了它们直接作用于翻译机构的假设。这些化合物是第一批能够调节起始密码子识别的保真度的化合物。我们建议继续这一筛选，以寻找对起始密码子识别的保真度有更强影响的其他化合物。与MLPCN中心之一的合作将极大地提高这项工作的效率。此外，这些中心的化学合成专业知识将使人们能够执行详细的结构-活性关系，以及优化已发现的活性化合物的功效。除了作为探索起始密码子识别的分子力学工具的固有效用外，调节这一过程保真度的化合物将是新的抗增殖剂、抗真菌和抗寄生虫剂以及改善起始密码子突变引起的遗传病的治疗方法的有价值的线索。

DESCRIPTION (provided by applicant): The recognition of the start codon by the eukaryotic translational machinery is a critical step in gene expression. It sets the reading frame in which the mRNA will be decoded and defines the N-terminus of the corresponding protein. We have developed an in vivo high-throughput dual luciferase assay that allows accurate measurement of the fidelity of start codon recognition in S. cerevisiae. Using this assay and a powerful secondary assay that eliminates agents that generally affect expression or activity of one or both luciferase enzyme, we screened over 50,000 molecules for compounds that reduce the fidelity of start codon recognition. From this screen two structurally similar molecules were found that increase use of near-cognate codons (e.g., UUG, CUG, etc.) as initiation sites. These activities were confirmed in several tertiary assays. Further in vivo analysis of the effects of the compounds supported the hypothesis that they act directly on the translational apparatus. These are the first compounds to be identified that modulate the fidelity of start codon recognition. We propose to continue this screen to find additional compounds with stronger effects on the fidelity of start codon recognition. Collaboration with one of the MLPCN centers will dramatically increase the efficiency of this undertaking. In addition, the chemical synthesis expertise of the centers will enable detailed structure-activity relationships to be performed, as well as optimization of the efficacy of active compounds that are discovered. In addition to their inherent utility as tools for probing the molecular mechanics of start codon recognition, compounds that modulate the fidelity of this process would be valuable leads for new antiproliferative agents, antifungal and antiparasitic agents, and therapeutics to ameliorate genetic diseases caused by initiation codon mutations.