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

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

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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）：所有使者RNA（mRNA）必须经历成熟或处理事件以定义其3'端，在添加长多腺苷酸化尾巴之前。没有这两个事件，mRNA无法翻译并被摧毁。因此，该过程称为裂解和聚腺苷酸化，是所有mRNA的必要事件，并由称为裂解和聚腺苷酸化机制的蛋白质组成。最近和挑衅性的证据表明，随着细胞的发生，它们表达了较短的信使RNA。这种缩短是由于被称为替代裂解和聚腺苷酸化的过程的结果，在这种过程中，细胞将mRNA的3'末端的位置更改为更近端的位点。癌细胞中该过程的方式被误导的机制知之甚少，但很可能涉及裂解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poly（a）位点选择的高通量筛选测定法，以识别抑制近端聚（a）位点使用的小分子。该测定法将基于在我的实验室中开发的非常成功的转录记者，以监测3'端形成反应的效率。与Te William S. Dunn Chemical Genomics筛查设施主管Clifford Stephan博士合作，我们将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个独特的分子）的效果。最后，我们将开发截面以确定抑制剂的特异性以及三个独立的二级筛选，以区分直接与间接抑制裂解和聚腺苷酸化的抑制作用，以及使用大量平行测序方案的阳性化合物的全球影响。这些化合物将有助于审问执行此过程的复合物的功能，并且也可能是潜在的铅化合物来开发能够抑制癌细胞中这种过程的“智能”化学治疗剂。