High Throughput Screen for Myotonic Dystrophy Type 1

描述（由申请人提供）：强直性肌营养不良症（DM 1）是人类最常见的成人型肌营养不良症。目前，没有治愈或FDA批准的药物用于DM 1和相关疾病。DM 1的分子基础是蛋白激酶基因DMPK的3'非翻译区中CTG重复序列的扩增。这种缺陷导致编码扩展的CUG重复序列（CUGexp）的突变体DMPK RNA的表达，所述CUG重复序列形成大的核内RNA-蛋白质复合物或病灶。CUGexp RNA的表达导致异常的RNA剪接，这反过来又与DM 1病理学的关键特征的发展有关。我们假设，在DM 1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逆转DM 1病理。为了验证这一假设，我们开发了一种初级HTS和二级命中验证测定，以鉴定改变CUGexp RNA生物学而不影响正常转录物的小分子。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种高度多样化的小分子组成，代表了超过一百万种化合物的库。从该文库中初步筛选了2，500个小分子，鉴定出一种有效的先导化合物MDI 16，它可以逆转DM 1患者成肌细胞和DM 1的HSALR小鼠模型中的异常RNA剪接模式。在一个共同努力，以确定其他有效的铅化合物，我们提出了以下建议：目的1。在MLPCN中心实施初级HTS和次级命中确认试验。目标2.在基于三级细胞的测定中进行测试命中，以鉴定逆转四种关键细胞DM 1表型的高效分子。目标3。表征先导化合物的选择性和毒性，并使用实验室开发的一套基于细胞的测定法在细胞水平上确定其作用机制。目标4。与MLPCN中心一起，我们将以非粘附方式改进先导化合物的化学结构，以优化药理学特性并建立结构-活性关系。 1型强直性肌营养不良是一种神经肌肉疾病，目前尚无治疗方法。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。我们已经开发了一种灵敏的高通量筛选（HTS）来鉴定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，在DM 1患者成肌细胞和DM 1小鼠模型中挽救DM 1病理。由于我们的筛选已被证明可以鉴定出拯救DM 1病理的有效分子，因此在本申请中，我们提出通过用我们的HTS筛选MLPCN化学文库来鉴定其他先导化合物。多个导联的鉴定将大大提高DM 1的小分子治疗的可能性。

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.