High-throughput screen for inhibitors of human spliceosomes

描述(申请人提供)：前-信使核糖核酸剪接是去除非编码内含子，中断大多数基因转录，在真核基因表达中起着至关重要的作用。此外，剪接现在被广泛认为是蛋白质组复杂性的关键介体，因为它调节了同一转录物中不同外显子的替代包含。理解进行剪接的分子机制很重要，因为影响结构性剪接和选择性剪接的突变与包括癌症在内的许多人类疾病有关。这种机制被称为剪接体，是一个由5个结构RNA和100多个单独的多肽组成的大型蛋白质/RNA大分子复合体。剪接体通过一系列尚未完全表征的结构中间体来组装和发挥功能。剪接体的动态复杂性长期以来给详细的生化和结构研究带来了挑战。为了应对这一挑战，需要小分子抑制剂作为工具，它们将在剪接途径的不同步骤阻止剪接体。有剪接抑制剂的报道，但它们作为结构功能工具的用途尚未实现。开发一系列针对剪接体的有效小分子抑制剂对于剖析剪接体的机制和在疾病情况下操纵其功能至关重要。我们建议开发一套适用于潜在剪接体抑制剂的高通量筛选(HTS)的补充分析方法。一种测试将在体外环境下测试剪接，同时针对所有关键的剪接体蛋白/相互作用。一种互补的检测方法将监测酿酒酵母体内的剪接，并利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。与加州大学伯克利分校化学系的Scott Lokey博士合作，我们将使用这些分析方法筛选几个小分子文库，总计超过55,000种化合物。通过任何一种筛选确定的候选抑制剂将通过在人和酵母核提取液中建立的基于凝胶的分析来表征，以确定它们在剪接体动态功能的哪个点发挥作用。通过这些筛选确定的抑制剂将成为捕获剪接体的新工具，用于进一步的结构和生化研究。这些化合物还将作为先导化合物，用于开发针对剪接功能的药物。 公共卫生相关性：编辑大多数人类基因产物(称为剪接体)的细胞机制的改变与包括癌症在内的许多人类疾病有关。识别阻止这种细胞机械功能的化学物质将提供对剪接体工作原理的洞察，并可能成为治疗疾病的药物的先导。

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.