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

描述（由申请人提供）：MicroRNAs （miRNAs）是单链非编码rna，代表了最近发现的一类新的基因调控因子。据估计，每个miRNA控制着数百个基因靶点，并且miRNA参与了约30%的基因和几乎所有遗传途径的调控。有证据表明，它们在发育过程和病毒感染中发挥重要作用，并可作为致癌基因和肿瘤抑制基因。因此，有人认为miRNA和miRNA通路的组成部分可以作为发现新型治疗剂的靶点。尽管miRNA通路在生理和病理过程中很重要，如癌症的发展，但据我们所知，尚未发现该通路的小分子抑制剂。为了填补这一空白，将开发一种高通量的基于细胞的检测方法，用于筛选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)将融合到人miRNA-21结合位点的荧光素酶基因（miR- 21）组成的报告系统引入人HeLa癌细胞。小分子抑制miRNA通路将导致荧光素酶报告信号的增加，从而提供有利的正读出。该检测将用于高通量筛选小分子文库（约1000个化合物），以发现干扰miRNA通路的分子。2)将开发三个二级分析来验证和表征来自主筛选的化合物命中。这些检测将排除非特异性小分子撞击，并提供活性化合物靶向的miRNA途径步骤的更详细图像。通过第二代化合物阵列的合成，将进一步研究和改进正命中。所获得的信息将最终导致阐明一个特定的目标-小分子相互作用。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。由于miRNA参与多种人类疾病（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，我们在筛选中发现的小分子有望对人类健康产生广泛的影响。这些分子将成为开发新的生物工具和新的治疗剂的有希望的先导化合物。例如，针对特定mirna的化学抑制剂，如临床相关的miR-21，有可能被开发成新的抗癌疗法。由于mirna在癌症和病毒感染等几种人类疾病中发挥着关键作用，我们在筛选中发现的小分子有望对人类健康产生广泛的影响。这些分子将成为生物医学研究中开发新的化学工具和新的治疗药物的有希望的先导化合物。针对特定mirna的小分子抑制剂，如临床相关的miR-21，有可能发展成为新的癌症治疗药物。

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.