High-Throughput Assay for the Discovery of Small Molecule Inhibitors of microRNA

用于发现 microRNA 小分子抑制剂的高通量检测

基本信息

批准号：
8299685
负责人：
Alexander Deiters
金额：
$ 3.67万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-30 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8299685
关键词：
Animal Model Animals Antineoplastic Agents Antisense Oligonucleotides Apoptotic Binding Biogenesis Biological Biological Assay Cell Differentiation process Cell Line Cell Proliferation Cell physiology Cells Chemicals Data Detection Development Disease Down-Regulation Drug Delivery Systems Exhibits Future Generations Genes Genetic Genetic Transcription Goals HIV Health Hepatitis C Hepatitis C virus Human Human Genome Human Pathology Individual Investigation Knowledge Lead Link Location Luciferases Malignant Neoplasms Messenger RNA MicroRNAs Molecular Molecular Profiling Nature Nucleotides Oligonucleotides Pathway interactions Pharmaceutical Preparations Phenotype Post-Transcriptional Regulation Process Regulation Regulator Genes Reporter Research Research Personnel Specific qualifier value Specificity Structure Testing Therapeutic Time Tissues Transcription Process Transfection Untranslated Regions Up-Regulation Validation Viral Cancer Virus Diseases Virus Replication assay development base cancer type chemotherapeutic agent genetic manipulation high throughput screening human disease improved inhibitor/antagonist innovation interest luminescence new therapeutic target novel novel strategies outcome forecast public health relevance small molecule stable cell line stem tool tumor tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are short, single-stranded RNAs of 21-23 nucleotides that are enzymatically processed from stem-loop precursors encoded within the human genome. They bind to untranslated regions in messenger RNA and induce a down-regulation of their transcription. It has been estimated that approximately 1000 microRNAs exist in humans, which control up to 30% of all genes. Thus, it is not surprising that the misregulation (either up- or down-regulation) of certain microRNAs has been linked to the development and prognosis of many types of cancer and other human diseases, including viral infections. The microRNA miR-122 and miR-155 have been recognized as important miRNAs involved in Hepatitis C Virus infection and cancer development. Although the connections between those miRNAs and human diseases have been made, very little is known about the biogenesis and regulation of individual microRNAs in healthy tissue, and what causes their misregulation in diseased tissue. Small molecule inhibitors of miRNA function will be unique pharmacological probes to close this knowledge gap. In contrast to the commonly used oligonucleotide antisense agents to inhibit miRNA function through hybridization and duplex formation, small molecule inhibitors can interfere with any step of the miRNA pathway. Thus, they can reveal important information about transcriptional and post-transcriptional regulation of particular miRNAs. Moreover, small molecule miRNA modulators have significant advantages over oligonucleotide antisense agents: they can be easily shared; they are more stable intracellularly; they are easily delivered into cells, animals, and humans; timing and location of delivery can be controlled; and they can be directly used on various cell lines and in different model organisms. We are proposing to develop a cell-based high-throughput assay using luminescence readouts for the discovery of small molecule inhibitors of miR-122 and miR-155. In addition to establishing the primary assay, a set of three secondary assays will be developed to validate and characterize the compound hits from the primary screen. These assays will exclude non-specific small molecule hits and deliver a more detailed picture of the activity and specificity of the identified compounds. Specifically, we will achieve this goal via the following aims: ' Specific Aim 1: Assay development for miR-122 and miR-155 small molecule inhibitors. This will be accomplished through the following sub-aims: (1) Build luciferase reporter constructs for the intracellular detection of miR-122 and miR-155 function. (2) Test the reporters in cells via transient transfection and determine the parameter Z' using antagomir antisense agents. ' Specific Aim 2: Assay configuration for high-throughput screening of miR-122 and miR-155 small molecule inhibitors. This will be accomplished through the following sub-aims: (1) Modify reporter constructs for the generation of stable cell lines. (2) Generate stable cell lines expressing miR-122 and miR-155 reporters. (3) Test the stable cell lines with antagomir antisense agents and determine the parameter Z'. (4) Conduct a pilot screen of 1364 compounds. (5) Establish secondary assays to eliminate hit compounds that are not miRNA-specific inhibitors and to validate hit compounds. Based on our expertise in developing the first small molecule inhibitor of miRNA function, specifically of miR- 21, and based on the substantial preliminary data presented, we do not expect any difficulties in achieving the two aims stated above. Our long term goal is to develop chemical tools to better understand the molecular mechanisms of miRNA biogenesis, of the functions of specific miRNAs involved in human disease, and to assess the global impact of miRNAs on various cellular processes and pathways. The small molecules that will be discovered from a high- throughput screen at the MLPCN are expected to have a broad impact on human health, due to the involvement of miRNAs in several human pathologies (including cancer and viral infection) and the increasing interest in the miRNA pathway as a drug target. The establishment of miRNAs as molecular drug targets together with novel small molecule inhibitors has the potential to provide a paradigm changing effect on the discovery of targeted chemotherapeutic agents. Furthermore, the developed inhibitors will be used as innovative and highly specific chemical tools for the study of the biogenesis and function of the targeted miRNAs. PUBLIC HEALTH RELEVANCE: The miRNAs miR-122 and miR-155 are involved in hepatitis C virus (HCV) replication and cancer manifestation. A high-throughput assay for small molecule inhibitors of these miRNAs together with a set of secondary assays will be developed. Discovered small molecules will be unique probes for the detailed investigation of the regulation and biogenesis of these disease-relevant miRNAs, and have the potential to validate both miRNAs as fundamentally novel therapeutic targets.

描述（由申请人提供）：microRNA（miRNA）是21-23个核苷酸的短，单链的RNA，这些RNA是从人类基因组中编码的茎环前体进行酶促处理的。它们与信使RNA中未翻译的区域结合，并诱导其转录下调。据估计，人类中存在大约1000个microRNA，其中控制着所有基因的30％。因此，毫不奇怪，某些microRNA的正调（上调或下调）与许多类型的癌症和其他人类疾病（包括病毒感染）的发展和预后有关。 MicroRNA miR-122和miR-155被认为是参与乙型肝炎病毒感染和癌症发育的重要miRNA。尽管已经建立了这些miRNA和人类疾病之间的联系，但对健康组织中单个microRNA的生物发生和调节却鲜为人知，以及导致其在患病组织中的正不调节的原因。 miRNA功能的小分子抑制剂将是缩小该知识差距的独特药理探针。与常用的寡核苷酸反义剂通过杂交和双链形成抑制miRNA功能的反义剂，小分子抑制剂可以干扰miRNA途径的任何步骤。因此，他们可以揭示有关特定miRNA的转录和转录后调节的重要信息。此外，小分子miRNA调节剂比寡核苷酸反义剂具有显着的优势：它们很容易共享；它们在细胞内更稳定；它们很容易被输送到细胞，动物和人类中。可以控制交货的时间和位置；它们可以直接用于各种细胞系和不同的模型生物。我们建议使用发光读数开发基于细胞的高通量测定法，以发现miR-122和miR-155的小分子抑制剂。除了建立主要测定法外，还将开发一组三个辅助测定，以验证和表征主屏幕上的复合命中。这些测定法将排除非特异性的小分子命中，并提供有关已识别化合物的活性和特异性的更详细图片。具体而言，我们将通过以下目标实现此目标：“特定目标1：MiR-122和miR-155小分子抑制剂的测定开发。这将通过以下子AIMS来实现：（1）建立荧光素酶报告基因构建体，以用于miR-122和miR-155功能的细胞内检测。（2）通过瞬时转染测试细胞中的报告者，并使用Antagomir反义剂确定参数Z'。 '特定目标2：用于对miR-122和miR-155小分子抑制剂进行高通量筛选的测定构型。这将通过以下子AIMS来完成：（1）修改记者构造，以生成稳定的细胞系。（2）产生表达miR-122和miR-155记者的稳定细胞系。（3）用Antagomir反义剂测试稳定的细胞系，并确定参数Z'。（4）进行1364种化合物的试验屏幕。（5）建立次要测定，以消除不是miRNA特异性抑制剂并验证命中化合物的命中化合物。基于我们在开发miRNA功能的第一个小分子抑制剂方面的专业知识，特别是miR-21的抑制剂，并基于提出的大量初步数据，我们期望实现上述两个目标时都没有任何困难。我们的长期目标是开发化学工具，以更好地了解miRNA生物发生的分子机制，涉及人类疾病的特定miRNA的功能，并评估miRNA对各种细胞过程和途径的全球影响。由于miRNA参与了几种人类病理（包括癌症和病毒感染），并且对miRNA途径的兴趣增加，预计将从MLPCN处的高吞吐量筛查中发现的小分子对人类健康产生广泛的影响。将miRNA作为分子药物靶标以及新型的小分子抑制剂的建立可能会对发现靶向化学治疗剂的发现提供范式变化的影响。此外，开发的抑制剂将用作研究靶向miRNA的生物发生和功能的创新和高度特定的化学工具。公共卫生相关性：miRNA miR-122和miR-155参与丙型肝炎病毒（HCV）复制和癌症表现。将开发针对这些miRNA的小分子抑制剂以及一组次级测定的高通量测定法。发现的小分子将是对这些与疾病相关的miRNA的调节和生物发生的详细研究的独特探针，并有可能验证这两个miRNA作为从根本上新颖的治疗靶标。