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

描述（由申请人提供）：MicroRNAs （miRNAs）是短的单链rna，由21-23个核苷酸组成，由人类基因组编码的茎环前体酶促加工而成。它们结合到信使RNA的非翻译区域并诱导其转录下调。据估计，人类体内大约存在1000个microrna，它们控制着30%的基因。因此，某些microrna的失调（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，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microrna与人类疾病之间的联系已经确定，但对于健康组织中单个microrna的生物发生和调控，以及它们在病变组织中失调的原因，人们知之甚少。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。与常用的寡核苷酸反义药物通过杂交和双工形成抑制miRNA功能不同，小分子抑制剂可以干扰miRNA通路的任何步骤。因此，它们可以揭示特定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著的优势：它们可以很容易地共享；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；可控制交货时间和地点；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测定方法，使用发光读数来发现miR-122和miR-155的小分子抑制剂。除了建立初级分析外，还将开发一套三种次级分析来验证和表征初级筛选的化合物命中。这些检测将排除非特异性的小分子撞击，并提供鉴定化合物的活性和特异性的更详细的图片。具体来说，我们将通过以下目标实现这一目标：“特定目标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)构建荧光素酶报告结构，用于细胞内检测miR-122和miR-155的功能。(2)瞬时转染细胞检测报告基因，并使用安塔戈米尔反义剂测定参数Z'。特异性目标2：高通量筛选miR-122和miR-155小分子抑制剂的检测配置。这将通过以下子目标来实现：(1)修改报告基因构建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)用安塔戈莫反义剂检测稳定细胞系，确定参数Z′。(4)对1364种化合物进行试点筛选。(5)建立二级分析，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miRNA功能（特别是miR- 21）的第一个小分子抑制剂方面的专业知识，以及基于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，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。由于miRNA参与多种人类病理（包括癌症和病毒感染），以及人们对miRNA途径作为药物靶点的兴趣日益增加，MLPCN将通过高通量筛选发现的小分子有望对人类健康产生广泛影响。mirna与新型小分子抑制剂一起作为分子药物靶点的建立，有可能为靶向化疗药物的发现提供一种改变范式的作用。此外，开发的抑制剂将被用作研究靶向mirna的生物发生和功能的创新和高度特异性的化学工具。

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.