Modulators of the fidelity of start codon recognition in eukaryotes

描述（由申请人提供）：真核生物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被解码的阅读框，并定义了相应蛋白质的n端。我们开发了一种体内高通量双荧光素酶测定方法，可以精确测量酿酒酵母开始密码子识别的保真度。使用该分析和强大的二级分析，消除了通常影响一种或两种荧光素酶的表达或活性的试剂，我们筛选了超过50,000个分子，以降低起始密码子识别的保真度。从这个筛选中发现两个结构相似的分子增加了近同源密码子（例如，UUG， CUG等）作为起始位点的使用。这些活性在几次三级分析中得到证实。进一步的体内分析化合物的影响支持假设，他们直接作用于翻译装置。这些是第一批被鉴定的调节起始密码子识别保真度的化合物。我们建议继续进行筛选，以找到对起始密码子识别保真度有更强影响的其他化合物。与其中一个MLPCN中心的合作将大大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详细的结构-活性关系得以执行，并优化所发现的活性化合物的功效。除了作为探测起始密码子识别的分子机制的固有工具外，调节这一过程的保真度的化合物将成为新的抗增殖剂，抗真菌和抗寄生虫剂以及改善由起始密码子突变引起的遗传疾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. PUBLIC HEALTH RELEVANCE: We propose to screen for molecules that change how accurately the eukaryotic protein synthesis machinery finds the appropriate place to begin decoding a messenger RNA to make the corresponding protein. Molecules with this activity could be developed into new drugs to treat cancers and a variety of other diseases.