A Cell-based Screen for Small Molecule Modulators of the miRNA Pathway

基于细胞的 miRNA 通路小分子调节剂筛选

• 批准号: 7291173
• 负责人: QIHONG HUANG
• 金额: $ 9.51万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-05 至 2009-08-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7291173
• 关键词: Affinity; Animal Model; Antiviral Agents; Area; Binding; Binding Sites; Biogenesis; Biological; Biological Assay; Biomedical Research; Cell physiology; Cells; Chemicals; Class; Collection; Complementary DNA; Development; Developmental Process; Disruption; Drug Delivery Systems; Eligibility Determination; Evaluation; Gene Targeting; Generations; Genes; Genetic; Goals; Health; Hela Cells; Homeostasis; Human; Knowledge; Lead; Libraries; Luc Gene; Luciferases; Malignant Neoplasms; MicroRNAs; Molecular; Molecular Weight; Pathologic Processes; Pathway interactions; Phenotype; Physiological; Play; Positioning Attribute; Process; Property; Proteins; RNA Interference; Range; Reading; Recording of previous events; Regulation; Regulator Genes; Reporter; Research Personnel; Role; Screening procedure; Signal Transduction; Small Interfering RNA; Staging; Structure; Structure-Activity Relationship; System; Therapeutic; Therapeutic Agents; Tumor Suppressor Genes; United States Food and Drug Administration; Untranslated RNA; Validation; Viral Cancer; Virus Diseases; anti-cancer therapeutic; assay development; base; cancer cell; cell type; clinically relevant; design; genetic manipulation; high throughput screening; human disease; improved; inhibitor/antagonist; innovation; interest; member; multidisciplinary; novel; novel therapeutics; small hairpin RNA; small molecule; small molecule libraries; tool; tumor

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are single-stranded noncoding RNAs and represent a novel class of recently discovered gene regulators. It is estimated that each miRNA controls hundreds of gene targets and that miRNAs are involved in the regulation of about 30% of all genes and almost every genetic pathway. Evidence suggests that they play important roles in developmental processes and viral infection, and they can function as oncogenes and tumor suppressors. Hence, it has been suggested that miRNAs and the components of the miRNA pathway can serve as targets for the discovery of novel therapeutic agents. Although the miRNA pathway is important in physiological and pathological processes, such as cancer development, to our knowledge, no small molecule inhibitor of this pathway has been identified. To fill this gap, a high-throughput cell-based assay will be developed for the screening of small molecule modulators of the miRNA pathway. This goal will be achieved through the following two specific aims: 1) a reporter system consisting of a luciferase gene fused to the binding site of human miRNA-21 (miR- 21) will be introduced into human HeLa cancer cells. Inhibition of the miRNA pathway by small molecules will lead to an increase of a luciferase reporter signal and thus provide an advantageous positive read-out. This assay will be used to screen small molecule libraries (>1000 compounds) in a high throughput fashion to discover molecules that interfere with the miRNA pathway. 2) 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 miRNA pathway steps that are targeted by active compounds. Positive hits will be further investigated and improved through structure-activity relationship studies, via synthesis of second generation compound arrays. The information gained will ultimately lead to the elucidation of a specific target-small molecule interaction. Our long term goal is to develop chemical tools to better understand the molecular mechanisms of the miRNA biogenesis, the functions of specific miRNAs, and to assess the global impact of miRNAs on various cellular processes and pathways. Small molecules discovered in our screen are expected to have a broad impact on human health, due to the involvement of miRNAs in several human diseases (including cancer and viral infection) and the increasing interest in the miRNA pathway as a drug target. These molecules will be promising lead compounds for the development of new biological tools and new therapeutic agents. For example, chemical inhibitors against particular miRNAs, such as the clinically relevant miR-21, have the potential to be developed into novel anticancer therapeutics. Small molecules discovered in our screen are expected to have a broad impact on human health, due to the critical roles that miRNAs play in several human diseases such as cancer and viral infection. These molecules will be promising lead compounds for the development of new chemical tools in biomedical research and new therapeutic agents. Small molecule inhibitors against particular miRNAs, such as the clinically relevant miR-21, have the potential to be developed into novel cancer therapeutics.

描述（由申请人提供）：微小RNA（miRNA）是单链非编码RNA，代表了一类新发现的基因调控因子。据估计，每个miRNA控制数百个基因靶标，并且miRNA参与约30%的所有基因和几乎所有遗传途径的调节。有证据表明，它们在发育过程和病毒感染中起重要作用，并且它们可以作为癌基因和肿瘤抑制因子发挥作用。因此，已经表明miRNA和miRNA途径的组分可以作为发现新治疗剂的靶标。尽管miRNA通路在生理和病理过程中很重要，例如癌症发展，但据我们所知，尚未鉴定出该通路的小分子抑制剂。为了填补这一空白，将开发一种基于细胞的高通量测定方法来筛选miRNA途径的小分子调节剂。这一目标将通过以下两个具体目标来实现：1）将由融合到人miRNA-21（miR- 21）的结合位点的荧光素酶基因组成的报告系统引入人HeLa癌细胞中。小分子对miRNA途径的抑制将导致荧光素酶报告信号的增加，从而提供有利的阳性读出。该测定将用于以高通量方式筛选小分子文库（>1000种化合物），以发现干扰miRNA途径的分子。2)将开发三种二次试验，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，并提供活性化合物靶向的miRNA途径步骤的更详细的图片。正命中将通过结构-活性关系研究，通过合成第二代化合物阵列进一步研究和改进。所获得的信息将最终导致特定靶标-小分子相互作用的阐明。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，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。在我们的筛选中发现的小分子预计将对人类健康产生广泛的影响，这是由于miRNA参与了几种人类疾病（包括癌症和病毒感染），并且对miRNA途径作为药物靶标的兴趣越来越大。这些分子将是开发新的生物工具和新的治疗药物的有前途的先导化合物。例如，针对特定miRNA（如临床相关的miR-21）的化学抑制剂具有开发成新型抗癌疗法的潜力。在我们的筛选中发现的小分子预计将对人类健康产生广泛的影响，因为miRNAs在癌症和病毒感染等多种人类疾病中发挥着关键作用。这些分子将是有前途的先导化合物，用于开发生物医学研究中的新化学工具和新的治疗剂。针对特定miRNA（如临床相关的miR-21）的小分子抑制剂具有开发成新型癌症治疗剂的潜力。