HTS to Identify Small Molecules Targeting Repeating Transcripts

描述（由申请人提供）：RNA在许多疾病中起着关键作用，因此可以作为重要的药物靶点。然而，绝大多数RNA靶标尚未被用于靶向研究。这是由于缺乏结合小分子的RNA基序的信息，例如，这些基序是内部和发夹环。我们提出的研究的总体目标是利用MLPCN资源来识别特异性结合重复1x1核苷酸AA内环的小分子，这些内环存在于导致亨廷顿病（HD）和脊髓小脑性共济失调3型（SCA3）的扩展r（CAG）重复序列中，目前尚无治愈方法。我们建议使用染料置换法完成MLPCN筛选，其中当小分子与RNA靶标结合并置换染料时，荧光发射增加。这种方法已经使用LOPAC库和NIH临床收集进行了验证；因此，它非常适合HTS活动。确定的配体将在各种二级分析中进行测试。具体来说，引线将被测试为：a.)RNA结合选择性；b)结合三联体重复转录本的蛋白质的位移；c.)前mrna剪接缺陷的纠正；d)抑制亨廷顿突变的翻译，这是HD的病原体。

DESCRIPTION (provided by applicant): RNA plays a critical role in a host of diseases and thus can serve as an important drug target. The vast majority of RNA targets, however, have not been utilized in targeting endeavors. This is due to a lack of information on the RNA motifs that bind small molecules, such motifs are internal and hairpin loops, for example. The overall goal of our proposed research is to utilize MLPCN resources to identify small molecules that specifically bind to the repeating 1x1 nucleotide AA internal loops that are present in the expanded r(CAG) repeats that cause Huntington's Disease (HD) and Spinocerebellar Ataxia Type 3 (SCA3), which have no known cures. We propose to complete MLPCN screens using a dye-displacement assay in which fluorescence emission increases when a small molecule binds to an RNA target and displaces the dye. This approach has been validated using the LOPAC library and the NIH clinical collection; thus, it is well suited for a HTS campaign. Identified ligands will then be tested in a variety of secondary assays. Specifically, leads will be tested fo: a.) RNA binding selectivity; b.) displacement of proteins that bind the triplet-repeating transcripts; c.) correction of pre-mRNA splicing defects; and d.) inhibition of translation of mutat Huntington, which is the causative agent in HD.