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

• 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
• 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

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）：微小RNA（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酶促加工而成。它们与信使RNA中的非翻译区结合并诱导其转录下调。据估计，人类中存在大约1000种microRNA，控制着高达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被认为是参与丙型肝炎病毒感染和癌症发展的重要miRNA。虽然这些miRNAs与人类疾病之间的联系已经建立，但对健康组织中单个microRNAs的生物发生和调节以及导致其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是填补这一知识空白的独特药理学探针。与通常使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反，小分子抑制剂可以干扰miRNA途径的任何步骤。因此，它们可以揭示关于特定miRNA的转录和转录后调控的重要信息。此外，小分子miRNA调节剂相对于寡核苷酸反义剂具有显著的优点：它们可以容易地共享;它们在细胞内更稳定;它们容易递送到细胞、动物和人类中;递送的时间和位置可以控制;并且它们可以直接用于各种细胞系和不同的模式生物体中。我们建议开发一种基于细胞的高通量检测方法，使用发光读数来发现miR-122和miR-155的小分子抑制剂。除了建立初步试验外，还将开发一组三种次要试验，以验证和表征初步筛选的化合物命中。这些测定将排除非特异性小分子命中，并提供所鉴定化合物的活性和特异性的更详细的图片。具体而言，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小分子抑制剂的测定开发。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Escheromir反义试剂确定参数Z'。 具体目标2：用于miR-122和miR-155小分子抑制剂的高通量筛选的测定配置。这将通过以下子目标来实现：（1）修饰报告基因构建体以产生稳定的细胞系。(2)产生表达miR-122和miR-155报告基因的稳定细胞系。(3)用Eschomir反义试剂测试稳定的细胞系，并确定参数Z '。(4)对1364种化合物进行中试筛选。(5)建立二级检测以消除不是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，以及基于所提供的大量初步数据，我们预计在实现上述两个目标方面不会遇到任何困难。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全球影响。预计将在MLPCN的高通量筛选中发现的小分子将对人类健康产生广泛影响，这是由于miRNA参与了几种人类病理学（包括癌症和病毒感染），并且对miRNA途径作为药物靶标的兴趣日益增加。作为分子药物靶标的miRNA与新型小分子抑制剂一起的建立具有在靶向化疗剂的发现上提供改变范例的作用的潜力。此外，开发的抑制剂将被用作创新的和高度特异性的化学工具，用于研究靶向miRNA的生物起源和功能。 公共卫生相关性：miRNAs miR-122和miR-155参与丙型肝炎病毒（HCV）复制和癌症表现。将开发这些miRNA的小分子抑制剂的高通量测定以及一组二级测定。发现的小分子将是详细研究这些疾病相关miRNAs的调控和生物发生的独特探针，并有可能验证这两种miRNAs作为全新的治疗靶点。