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 RNA，由人类基因组内编码的茎环前体经酶促加工而成。它们与信使 RNA 中的非翻译区域结合并诱导其转录下调。据估计，人类体内存在大约 1000 种 microRNA，它们控制着多达 30% 的基因。因此，某些 microRNA 的错误调节（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，这并不奇怪。 microRNA miR-122 和 miR-155 已被认为是参与丙型肝炎病毒感染和癌症发展的重要 miRNA。尽管这些 miRNA 与人类疾病之间的联系已经被确定，但人们对健康组织中单个 microRNA 的生物发生和调节以及导致它们在患病组织中失调的原因知之甚少。 miRNA 功能的小分子抑制剂将成为弥补这一知识空白的独特药理学探针。与常用的通过杂交和双链体形成来抑制 miRNA 功能的寡核苷酸反义试剂相比，小分子抑制剂可以干扰 miRNA 途径的任何步骤。因此，它们可以揭示有关特定 miRNA 的转录和转录后调控的重要信息。此外，小分子miRNA调节剂比寡核苷酸反义剂具有显着优势：它们可以轻松共享；它们在细胞内更稳定；它们很容易被输送到细胞、动物和人类体内；交货时间和地点可以控制；它们可以直接用于各种细胞系和不同的模式生物。我们提议开发一种基于细胞的高通量检测，使用发光读数来发现 miR-122 和 miR-155 的小分子抑制剂。除了建立主要测定之外，还将开发一组三个辅助测定来验证和表征来自初级筛选的化合物命中。这些测定将排除非特异性小分子命中，并提供所识别化合物的活性和特异性的更详细信息。具体来说，我们将通过以下目标实现这一目标：具体目标 1：miR-122 和 miR-155 小分子抑制剂的检测开发。这将通过以下子目标来实现：（1）构建用于细胞内检测 miR-122 和 miR-155 功能的荧光素酶报告基因构建体。 (2) 通过瞬时转染测试细胞中的报告基因，并使用 antagomir 反义试剂确定参数 Z'。具体目标 2：用于高通量筛选 miR-122 和 miR-155 小分子抑制剂的测定配置。这将通过以下子目标来实现：（1）修改报告基因构建体以产生稳定的细胞系。 (2) 生成表达 miR-122 和 miR-155 报告基因的稳定细胞系。 (3)用antagomir反义剂测试稳定细胞系并确定参数Z'。 (4) 对1364个化合物进行中试筛选。 (5) 建立二次测定以消除非 miRNA 特异性抑制剂的命中化合物并验证命中化合物。基于我们在开发第一个 miRNA 功能（特别是 miR-21）小分子抑制剂方面的专业知识，并基于所提供的大量初步数据，我们预计实现上述两个目标不会有任何困难。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、参与人类疾病的特定 miRNA 的功能，并评估 miRNA 对各种细胞过程和途径的整体影响。由于 miRNA 参与多种人类病理学（包括癌症和病毒感染），并且人们对 miRNA 的兴趣日益浓厚，MLPCN 通过高通量筛选发现的小分子预计将对人类健康产生广泛影响。 miRNA 通路作为药物靶点。 miRNA 作为分子药物靶点的建立与新型小分子抑制剂的结合有可能为靶向化疗药物的发现提供范式改变效应。此外，所开发的抑制剂将用作创新和高度特异性的化学工具，用于研究目标 miRNA 的生物发生和功能。公共健康相关性：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 的小分子抑制剂的高通量测定以及一系列二级测定。发现的小分子将成为详细研究这些疾病相关 miRNA 的调控和生物发生的独特探针，并有可能验证这两种 miRNA 作为根本性的新型治疗靶点。