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

描述(申请人提供)：microRNAs(MiRNAs)是21-23个核苷酸的单链短链RNA，由人类基因组中编码的茎环前体经过酶处理而成。它们与信使RNA中的未翻译区域结合，并诱导其转录下调。据估计，人类体内大约存在1000个microRNAs，它们控制着多达30%的所有基因。因此，某些microRNAs的错误调控(无论是上调还是下调)与许多类型癌症和包括病毒感染在内的其他人类疾病的发展和预后有关也就不足为奇了。MicroRNA miR-122和miR-155被认为是参与丙型肝炎病毒感染和肿瘤发生的重要miRNAs。虽然这些miRNAs与人类疾病之间的联系已经被建立，但对健康组织中单个microRNAs的生物发生和调控，以及是什么导致它们在患病组织中的调控不当，人们知之甚少。MiRNA功能的小分子抑制剂将成为缩小这一认识差距的独特药理探针。与通常使用的通过杂交和双链形成抑制miRNA功能的寡核苷酸反义药物不同，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miRNAs转录和转录后调控的重要信息。此外，与寡核苷酸反义药物相比，小分子miRNA调节剂具有显著的优势：它们易于共享；细胞内更稳定；它们易于输送到细胞、动物和人类体内；输送的时间和位置可以控制；它们可以直接用于各种细胞系和不同的模式生物。我们建议开发一种基于细胞的高通量分析方法，使用发光读数来发现miR-122和miR-155的小分子抑制剂。除了建立一次化验外，还将开发一套三种二次化验，以验证和表征来自一次筛查的化合物。这些分析将排除非特异性的小分子撞击，并提供已鉴定化合物的活性和特异性的更详细的图像。具体地说，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发展。这将通过以下子目标来实现：(1)构建用于检测miR-122和miR-155功能的胞内荧光素酶报告基因。(2)用瞬时转染法检测细胞内的报告基因，并用Anagomir反义剂测定参数Z‘。‘具体目标2：高通量筛选miR-122和miR-155小分子抑制剂的分析配置。这将通过以下子目标来实现：(1)为产生稳定的细胞系而修改报告构建体。(2)建立稳定表达miR-122和miR-155报告基因的细胞系。(3)用Anagomir反义核酸检测稳定的细胞株，并确定参数Z‘。(4)对1364个化合物进行试点筛选。(5)建立二次检测以消除非miRNA特异性抑制剂的HIT化合物，并验证HIT化合物。基于我们在开发第一个miRNA功能小分子抑制剂，特别是miR-21功能小分子抑制剂方面的专业知识，以及根据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，以及参与人类疾病的特定miRNAs的功能，并评估miRNAs对各种细胞过程和途径的全球影响。将在MLPCN的高通量筛查中发现的小分子预计将对人类健康产生广泛影响，这是因为miRNAs参与了几种人类病理(包括癌症和病毒感染)，而且人们对miRNA途径作为药物靶点的兴趣越来越大。MiRNAs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一种改变范式的效果。此外，所开发的抑制剂将被用作创新的和高度特异的化学工具，用于研究靶向miRNAs的生物发生和功能。 公共卫生相关性：miRNAs miR-122和miR-155与丙型肝炎病毒(丙型肝炎病毒)复制和癌症表现有关。将开发一种高通量的检测这些miRNAs的小分子抑制剂的方法，以及一套二次检测方法。已发现的小分子将是详细研究这些与疾病相关的miRNAs的调节和生物发生的独特探针，并有可能验证这两个miRNAs作为根本上新的治疗靶点。

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.