High-throughput screen for inhibitors of human spliceosomes

人类剪接体抑制剂的高通量筛选

• 批准号: 8212367
• 负责人: Melissa S Jurica
• 金额: $ 29.9万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8212367
• 关键词: Affect; Alternative Splicing; Asses; Biochemical; Biological Assay; Cell physiology; Chemicals; Chemistry; Collaborations; Collection; Complex; Disease; Drug Delivery Systems; Eukaryota; Excision; Exons; Foundations; Functional RNA; Gel; Gene Expression; Genes; Growth; Human; In Vitro; Individual; Introns; Lead; Libraries; Liquid substance; Macromolecular Complexes; Malignant Neoplasms; Mediator of activation protein; Messenger RNA; Molecular; Monitor; Mutation; Nuclear Extract; Pathway interactions; Pharmaceutical Preparations; Proteins; Proteome; RNA; RNA Splicing; Reporting; Reproducibility; Robot; Saccharomyces cerevisiae; Screening procedure; Solutions; Spliceosome Assembly Pathway; Spliceosomes; Structure; System; Testing; Transcript; Work; Yeasts; base; high throughput screening; human disease; in vivo; inhibitor/antagonist; insight; interest; mRNA Precursor; meetings; mutant; polypeptide; public health relevance; small molecule; small molecule libraries; tool

DESCRIPTION (provided by applicant): Pre-mRNA splicing is the removal of the non-coding introns that interrupt most gene transcripts and serves an essential step in eukaryotic gene expression. Furthermore, splicing is now widely recognized as a key mediator of proteome complexity by regulating alternative inclusion of different exons from the same transcript. It is important to understand the molecular machinery that carries out splicing because mutations that affect both constitutive and alternative splicing are associated with a number of human diseases, including cancers. The machinery, termed the spliceosome, is a large protein/RNA macromolecular complex comprised of five structural RNAs and over 100 individual polypeptides. The spliceosome assembles and functions via a progression of structural intermediates that are not yet fully characterized. The dynamic complexity of the spliceosome has long posed a challenge to detailed biochemical and structural studies. To meet this challenge, small molecule inhibitors that will arrest spliceosomes at different steps along the splicing pathway are needed as tools. There are reports of splicing inhibitors, but their utility as structure-function tools has not yet been realized. Developing an arsenal of potent small molecule inhibitors that target the spliceosome is critical for dissecting its mechanisms and for being able to manipulate its function in disease situations. We propose to develop a complementary set of assays suitable for high-throughput screening (HTS) for potential spliceosome inhibitors. One assay will test splicing in an in vitro setting and simultaneously target all critical spliceosome proteins/interactions. A complementary assay will monitor splicing in vivo in S. cerevisiae and use synthetic lethality with spliceosome mutants to target key steps of spliceosome function. In collaboration with Dr. Scott Lokey in the Chemistry Department at UCSC, we will use the assays to screen several small molecule libraries totaling over 55,000 compounds. Candidate inhibitors identified by either screen will be characterized by established gel-based assays in both human and yeast nuclear extracts to determine at which point of spliceosome dynamic function they exert their effects. Inhibitors identified by these screens will serve as new tools to trap spliceosome for further structural and biochemical studies. The compounds will also serve as lead compounds for developing drugs that target splicing function. PUBLIC HEALTH RELEVANCE: Alterations in the cellular machinery that edits most human gene products (termed spliceosomes) are associated with a number of human diseases, including cancers. Identifying chemicals that block the function of this cellular machinery will provide insights into the workings of spliceosomes and potentially serve as leads for drugs to treat disease.

描述（由申请人提供）：前mRNA剪接是去除中断大多数基因转录物的非编码内含子，并且在真核基因表达中是必不可少的一步。此外，通过调节来自同一转录物的不同外显子的替代性包含替代性，剪接现在被广泛认为是蛋白质复杂性的关键介体。重要的是要了解执行剪接的分子机制，因为影响组成型和替代剪接的突变与包括癌症在内的许多人类疾病有关。该机械称为剪接体，是一种大蛋白/RNA大分子复合物，由五个结构RNA和100​​多个单独的多肽组成。剪接体通过尚未完全表征的结构中间体的进展来组装和功能。长期以来，剪接体的动态复杂性对详细的生化和结构研究提出了挑战。为了应对这一挑战，需要将沿剪接途径的不同步骤阻止剪接体的小分子抑制剂作为工具。有关于剪接抑制剂的报道，但是它们作为结构功能工具的实用性尚未实现。开发靶向剪接体的有效小分子抑制剂的武库对于剖析其机制和能够在疾病情况下的功能而言至关重要。 我们建议开发一套适用于高通量筛查（HTS）的互补测定法，以实现潜在的剪接体抑制剂。一个测定法将在体外环境中测试剪接，并同时靶向所有关键的剪接蛋白/相互作用。补充测定法将在酿酒酵母中监测体内剪接，并使用剪接体突变体的合成致死性来靶向剪接体功能的关键步骤。通过与UCSC化学系的Scott Lokey博士合作，我们将使用这些测定法筛选几个小分子库，总计超过55,000种化合物。通过任何一个筛选确定的候选抑制剂将以人类和酵母核提取物中的基于凝胶的测定为特征，以确定它们在哪个夹层动态功能的位置发挥作用。这些筛选确定的抑制剂将作为捕获剪接体的新工具，以进行进一步的结构和生化研究。这些化合物还将用作开发靶向剪接功能的药物的铅化合物。 公共卫生相关性：编辑大多数人类基因产品（称为剪接体）的细胞机械的改变与包括癌症在内的许多人类疾病有关。识别阻断该细胞机械功能的化学物质将为剪接体的起作用提供见解，并有可能作为治疗疾病的药物的铅。