A HTS Assay for Inhibitors of Proximal Cleavage and Polyadenylation

近端切割和多聚腺苷酸化抑制剂的 HTS 测定

基本信息

批准号：
8624669
负责人：
ERIC J WAGNER
金额：
$ 27.36万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8624669
关键词：
Address Area Binding Sites Biochemical Bioinformatics Biological Assay Biological Models CCND1 gene Cancer Patient Cell Proliferation Cells Chemicals Collaborations Communities Complement Complex Consensus Data Databases Development Disease model Distal Elements Event Expressed Sequence Tags Gene Expression Genomics Goals Hand In Vitro Laboratories Lead Length Libraries Mantle Cell Lymphoma Massive Parallel Sequencing Mediating Messenger RNA MicroRNAs Molecular Monitor Normal Cell Organism Play Poly A Polyadenylation Positioning Attribute Process Property Protocols documentation Reaction Reporter Repression Research Resistance Role Sequence Analysis Site Small Interfering RNA Specificity Tail Technology Terminator Codon Testing Time Translating base cancer cell chemotherapeutic agent counterscreen deep sequencing design in vivo inhibitor/antagonist interest member miniaturize protein complex screening small molecule tool tumor progression tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): All messenger RNA (mRNA) must undergo a maturation or processing event to define their 3' end, which precedes the addition of a long polyadenylated tail. Without these two events, mRNAs fail to get translated and are destroyed. This process, termed cleavage and polyadenylation, is therefore a requisite event for all mRNA and is governed by a complex of proteins called the cleavage and polyadenylation machinery. Recent and provocative evidence suggests that as cells undergo tumorigenesis they express shorter messenger RNAs. This shortening is as a result of a process known as alternative cleavage and polyadenylation whereby the cell changes the position of the 3' terminus of the mRNA to a more proximal site. The mechanism of how this is process is misregulated in cancer cells is poorly understood but likely involves alterations in the properties of the cleavage and polyadenylation machinery or yet-to-be discovered members. Here, we propose to develop a highthroughput screening assay capable of monitoring poly(A) site choice in an effort to identify small molecules that inhibit use of the proximal poly(A) site. This assay will be based upon highly successful transcriptional readthrough reporters developed in my laboratory to monitor efficiency of 3' end formation reactions. In collaboration with Dr. Clifford Stephan, Director of te William S. Dunn Chemical Genomics Screening Facility, we will utilize this assay in chemical screens analyzing the effects of a highly diverse library of compounds (>50,000 unique molecules). Finally, we will develop counterscreens to determine the specificity of inhibitors as well as three independent secondary screens to distinguish direct versus indirect inhibition of cleavage and polyadenylation as well as the global impact of positive compounds using massively parallel sequencing protocols. These compounds will be instrumental to interrogate the function of the complex that performs this process and may also behave as potential lead compounds toward the development of "smart" chemotherapeutic agents capable of inhibiting this process in cancer cells.

描述（由申请人提供）：所有信使 RNA (mRNA) 都必须经历成熟或加工事件，以定义其 3' 末端，然后添加长聚腺苷酸尾。如果没有这两个事件，mRNA 就无法翻译并被破坏。因此，这个过程被称为裂解和聚腺苷酸化，是所有 mRNA 的必需事件，并且由称为裂解和聚腺苷酸化机制的蛋白质复合体控制。最近的令人兴奋的证据表明，当细胞发生肿瘤时，它们会表达更短的信使 RNA。这种缩短是由称为选择性切割和聚腺苷酸化的过程造成的，细胞通过该过程将 mRNA 3' 末端的位置改变到更近端的位点。癌细胞中这一过程如何被错误调节的机制尚不清楚，但可能涉及切割和聚腺苷酸化机制或尚未发现的成员的特性改变。在这里，我们建议开发一种能够监测聚腺苷酸位点选择的高通量筛选方法，以努力识别抑制近端聚腺苷酸位点使用的小分子。该测定将基于我的实验室开发的非常成功的转录通读报告器，用于监测 3' 末端形成反应的效率。我们将与 William S. Dunn 化学基因组筛选设施主任 Clifford Stephan 博士合作，在化学筛选中利用该测定法，分析高度多样化的化合物库（> 50,000 个独特分子）的影响。最后，我们将开发反筛选以确定抑制剂的特异性，以及三个独立的二级筛选，以区分切割和多聚腺苷酸化的直接抑制和间接抑制，以及使用大规模并行测序方案的阳性化合物的整体影响。这些化合物将有助于探究执行这一过程的复合物的功能，并且也可能作为潜在的先导化合物，用于开发能够抑制癌细胞中这一过程的“智能”化疗药物。