Modulators of the fidelity of start codon recognition in eukaryotes

真核生物起始密码子识别保真度的调节剂

• 批准号: 8326608
• 负责人: JON R. LORSCH
• 金额: $ 4.05万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8326608
• 关键词: Affect; Aftercare; Antifungal Agents; Antiparasitic Agents; Behavior; Biological Assay; Cells; Codon Nucleotides; Collaborations; Disease; Enzymes; Eukaryota; Fireflies; Firefly Luciferases; Gene Expression; Hereditary Disease; Initiator Codon; Life; Luciferases; Malignant Neoplasms; Measurement; Messenger RNA; Molecular Bank; Mutation; Pharmaceutical Preparations; Process; Production; Protein Biosynthesis; Proteins; Reading Frames; Relative (related person); Renilla Luciferases; Reporter; Saccharomyces cerevisiae; Screening procedure; Site; Structure; Structure-Activity Relationship; Therapeutic; Work; Yeasts; antiproliferative agents; chemical synthesis; high throughput screening; improved; in vivo; internal control; molecular mechanics; tool; vector

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.

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。它设置了将解码mRNA并定义相应蛋白质的N末端的阅读框。我们已经开发了一种体内高通量双荧光素酶测定法，该测定可以准确地测量酿酒酵母中起始密码子识别的保真度。使用该测定法和一个强大的次要测定，消除了通常影响一种或两种荧光素酶表达或活性的药物，我们筛选了超过50,000个分子的化合物，以降低起始密码子识别的保真度。从这个屏幕上发现了两个结构上相似的分子，可以增加使用近认知密码子（例如，UUG，CUG等）作为起始位点的使用。这些活动在几个三级测定中得到了证实。对化合物作用的进一步分析支持了它们直接在翻译设备上的假设。这些是要确定调节起始密码子识别的保真度的第一个化合物。我们建议继续此屏幕，以找到对起始密码子识别的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业务的效率。此外，这些中心的化学合成专业知识将使能够进行详细的结构活性关系，并优化发现的活性化合物的功效。除了它们作为探测起始密码子识别的分子力学的工具外，调节该过程的忠诚度的化合物对于新的抗增生剂，抗真菌和抗寄生虫，抗真菌和抗寄生虫和治疗剂以及由启动编码子突变引起的遗传性疾病的铅将是有价值的。