High Throughput Screen for Myotonic Dystrophy Type 1

1 型强直性肌营养不良的高通量筛查

• 批准号: 8209483
• 负责人: NOURI NEAMATI
• 金额: $ 4.08万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-22 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8209483
• 关键词: 3' Untranslated Regions; Adult; Affect; Antisense Oligonucleotides; Binding; Biological Assay; Biology; Cells; Chemical Structure; Consult; Data; Defect; Development; Disease; Ectopic Expression; FDA approved; Genes; Housing; Human; In Vitro; Lead; Libraries; Link; Machine Learning; Mediating; Molecular; Muscular Dystrophies; Myoblasts; Myotonic Dystrophy; Neuromuscular Diseases; Nuclear RNA; Pathology; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Probability; Property; Protein Family; Protein Kinase; Protein Kinase C Alpha; RNA; RNA Splicing; Screening procedure; Series; Structure-Activity Relationship; System; Testing; Therapeutic; Toxic effect; Transcript; Validation; base; high throughput screening; improved; in vivo; mouse model; mutant; protein complex; small molecule; small molecule libraries; therapeutic target; transcription factor

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM1) is the most common adult onset muscular dystrophy in humans. Currently, there is no cure or an FDA approved drug for DM1 and related diseases. The molecular basis of DM1 is the expansion of a CTG-repeat sequence in the 3' untranslated region of the protein kinase gene, DMPK. This defect results in the expression of mutant DMPK RNAs encoding expanded CUG repeats (CUGexp) that form large intra nuclear RNA-protein complexes or foci. Expression of CUGexp RNAs leads to abnormal RNA splicing, which in turn has been linked to the development of key features of DM1 pathology. We hypothesize that small molecules that degrade or disperse CUGexp RNAs in DM1 cells can re-establish normal splice patterns and reverse DM1 pathology. To test this hypothesis, we developed a primary HTS and a secondary hit validation assay to identify small-molecules that alter the biology of CUGexp RNAs without affecting the normal transcript. Our in house library was developed using a robust machine learning chemoinformatics platform and consists of 40,000 highly diverse small-molecules representing a library of over a million compounds. An initial screen of 2,500 small molecules from this library resulted in the identification of a potent lead compound, MDI16, which reverses aberrant RNA splice patterns in both DM1 patient myoblasts and in the HSALR mouse model for DM1. In a concerted effort to identify other potent lead compounds we propose the following: Aim 1. Implement primary HTS and the secondary hit validation assay in the MLPCN center. Aim 2. Test hits in tertiary cell-based assays to identify highly potent molecules that reverse four key cellular DM1 phenotypes. Aim 3. Characterize the selectivity and toxicity of lead compounds and identify their mechanism of action at the cellular level using a set of cell-based assays developed in the lab. Aim 4. In conjunction with the MLPCN center, we will refine the chemical structure of lead compounds reiteratively to optimize pharmacological properties and establish structure-activity relationships. PUBLIC HEALTH RELEVANCE: Lay Summary Myotonic dystrophy type 1 is a neuromuscular disorder for which there is no treatment or cure. Over the past few years exciting strides in our understanding of the mechanistic basis of this disorder have been made. Thus the field is poised to make a major breakthrough and develop a drug for this disorder. We have developed a sensitive high throughput screen (HTS) to identify compounds that cure or ameliorate pathologies associated with myotonic dystrophy. Currently we have discovered potent molecules, which rescue DM1 pathology in both DM1 patient myoblasts and in DM1 mouse models. As our screens have been proven to identify potent molecules that rescue DM1 pathology, in this application we propose to identify other lead compounds by screening the MLPCN chemical library with our HTS. Identification of multiple leads will greatly improve the probability of a small molecule therapy for DM1.

描述（由申请人提供）：肌发育症（DM1）是人类最常见的成年发作性肌营养不良症。目前，尚无治愈方法或FDA批准的DM1和相关疾病的药物。 DM1的分子基础是在蛋白激酶基因DMPK的3'未翻译区域中CTG重复序列的扩展。这种缺陷导致编码膨胀的CUG重复序列（CugeXP）的突变型DMPK RNA表达，该突变体形成了大型核内RNA-蛋白质复合物或焦点。 CugeXP RNA的表达导致异常RNA剪接，这又与DM1病理学的关键特征的发展有关。我们假设在DM1细胞中降解或分散CugeXP RNA的小分子可以重新建立正常的剪接模式并反向DM1病理学。为了检验这一假设，我们开发了主要的HTS和二级验证测定法，以识别改变CugeXP RNA生物学的小分子而不会影响正常转录本。我们的内部图书馆是使用强大的机器学习化学信息授权平台开发的，由40,000个高度多样的小分子组成，代表一个超过100万种化合物的库。该库中2,500个小分子的初始筛选导致鉴定有效的铅化合物MDI16，该化合物MDI16逆转了DM1患者肌细胞和HSALR小鼠模型中DM1的异常RNA剪接模式。为了确定其他有效的铅化合物，我们提出了以下方面的努力：AIM 1。在MLPCN中心实施主要HTS和次级命中验证测定法。 AIM 2。基于三级细胞的测定中的测试命中，以鉴定逆转四个键的细胞DM1表型的高效分子。 AIM 3。表征铅化合物的选择性和毒性，并使用实验室中开发的一组基于细胞的测定方法在细胞水平上确定其作用机理。 AIM 4。与MLPCN中心结合使用，我们将重新完善铅化合物的化学结构，以优化药理特性并建立结构活性关系。 公共卫生相关性：Lay摘要肌发育症1型是一种神经肌肉疾病，无法治愈或治愈。在过去的几年中，我们对这种疾病的机械基础的理解令人兴奋。因此，该领域有望取得重大突破并为这种疾病开发药物。我们已经开发了一种敏感的高吞吐量筛选（HTS），以识别治愈或改善与肌营养不良症相关的病理的化合物。目前，我们发现了有效的分子，这些分子在DM1患者肌细胞和DM1小鼠模型中挽救了DM1病理学。由于我们的筛选已被证明可以识别挽救DM1病理学的有效分子，因此在本应用中，我们建议通过使用HTS筛选MLPCN化学库来识别其他铅化合物。多个铅的识别将大大提高DM1小分子治疗的可能性。