Modulators of the fidelity of start codon recognition in eukaryotes

描述（由申请人提供）：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将在其中解码的阅读框架，并定义了相应蛋白质的N-末端。我们已经开发了一种体内高通量双荧光素酶测定法，可以准确测量S.酿酒。使用该测定和消除通常影响一种或两种荧光素酶的表达或活性的试剂的强大的二级测定，我们筛选了超过50，000个分子以寻找降低起始密码子识别保真度的化合物。从该筛选中发现了两种结构相似的分子，其增加了近同源密码子的使用（例如，UUG、CUG等）作为起始位点。这些活性在几项三级试验中得到证实。对化合物作用的进一步体内分析支持了它们直接作用于翻译装置的假设。这些是第一个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具有更强影响的其他化合物。与MLPCN中心之一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。除了它们作为用于探测起始密码子识别的分子机制的工具的固有效用之外，调节该过程的保真度的化合物将是用于新的抗增殖剂、抗真菌剂和抗寄生虫剂以及用于改善由起始密码子突变引起的遗传疾病的治疗剂的有价值的先导物。 公共卫生相关性：我们建议筛选分子，这些分子可以改变真核生物蛋白质合成机器找到合适位置的准确性，以便开始解码信使RNA，从而生成相应的蛋白质。具有这种活性的分子可以开发成治疗癌症和各种其他疾病的新药。

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.