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）：强直性肌营养不良症（DM1）是人类最常见的成人发病性肌营养不良症。目前，尚无针对 DM1 及相关疾病的治愈方法或 FDA 批准的药物。 DM1 的分子基础是蛋白激酶基因 DMPK 3' 非翻译区 CTG 重复序列的扩展。这种缺陷导致编码扩展 CUG 重复序列 (CUGexp) 的突变 DMPK RNA 的表达，从而形成大的核内 RNA-蛋白质复合物或焦点。 CUGexp RNA 的表达导致异常的 RNA 剪接，这反过来又与 DM1 病理学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 的小分子可以重建正常的剪接模式并逆转 DM1 病理。为了检验这一假设，我们开发了初级 HTS 和次级命中验证测定法，以识别改变 CUGexp RNA 生物学而不影响正常转录本的小分子。我们的内部库是使用强大的机器学习化学信息学平台开发的，由 40,000 个高度多样化的小分子组成，代表了超过一百万个化合物的库。对该文库中 2,500 个小分子的初步筛选结果鉴定出一种有效的先导化合物 MDI16，它可以逆转 DM1 患者成肌细胞和 DM1 HSALR 小鼠模型中的异常 RNA 剪接模式。为了共同努力识别其他有效的先导化合物，我们提出以下建议： 目标 1. 在 MLPCN 中心实施初级 HTS 和次级命中验证测定。目标 2. 在基于细胞的三级测定中测试命中结果，以鉴定可逆转四种关键细胞 DM1 表型的高效分子。目标 3. 使用实验室开发的一套基于细胞的测定方法表征先导化合物的选择性和毒性，并在细胞水平上确定其作用机制。目标4. 联合MLPCN中心，反复精炼先导化合物的化学结构，优化药理性质，建立构效关系。 公共卫生相关性：简单总结 1 型强直性肌营养不良是一种神经肌肉疾病，无法治疗或治愈。在过去的几年里，我们对这种疾病的机制基础的理解取得了令人兴奋的进展。因此，该领域有望取得重大突破并开发出治疗这种疾病的药物。我们开发了一种灵敏的高通量筛选（HTS）来识别能够治愈或改善与强直性肌营养不良相关的病理的化合物。目前，我们已经发现了有效的分子，可以挽救 DM1 患者成肌细胞和 DM1 小鼠模型中的 DM1 病理。由于我们的筛选已被证明可以识别拯救 DM1 病理的有效分子，因此在本应用中，我们建议通过使用我们的 HTS 筛选 MLPCN 化学库来识别其他先导化合物。多导联的鉴定将大大提高小分子治疗 DM1 的可能性。