Testing Compounds that Increase SMN levels for Efficacy in Mouse Models of SMA

描述（由申请人提供）：脊髓性肌萎缩症（SMA）是一种常染色体隐性形式的肌营养不良症，约1/6000的活产婴儿患病，临床表现为由于运动神经元退化导致的进行性肌无力。SMA是由运动神经元存活（SMN）蛋白水平不足引起的，通常是由于SMN 1基因的纯合缺失。一个几乎相同的拷贝基因SMN 2，不能保护SMA的发展，因为它的mRNA经历选择性剪接，产生一个截短的，不稳定的SMN蛋白。一小部分SMN 2转录物包含外显子7，编码与SMN 1相同的SMN蛋白。SMA的严重程度与SMN蛋白水平相关，SMN 2基因拷贝数低的婴儿在两年内死亡。患有轻度SMA的儿童有多个SMN 2拷贝。这种剂量依赖性通过上调SMN 2全长SMN蛋白的表达为SMA治疗创造了机会。我们实施了一项先导发现计划，以确定增加SMN蛋白水平的药物样化合物。使用一种新的基于细胞的报告分析，我们进行了高通量筛选（HTS）的大型化学多样性库。我们已经在SMN蛋白表达的二级测试中验证了多次命中的活性，并且目前正在通过一个集中的药物化学计划来优化三种化学上不同的支架。目标1将测试从这些结构-活性关系研究中出现的活性化合物的药物样性质、药代动力学和急性毒理学。目的2将在“7”SMA小鼠模型中检查最具活性和最合适的化合物，以确定化合物治疗是否增加体内SMN蛋白以及是否可以减轻疾病过程。目的3描述了一种组合方法来检查最有前途的化合物的功效。由于在基于细胞的HTS中鉴定的化合物似乎通过不同的机制刺激SMN水平，因此将通过评价这些先导化合物的成对组合的生物活性来采取最大化SMN诱导和最小化毒性的治疗方法。该提案合并了三个实验室的努力，这些实验室在SMA分子生物学、神经退行性疾病新疗法的开发以及SMA小鼠模型中药物试验的性能方面具有专业知识。该提案的预测结果是鉴定出可提高SMA模型小鼠中SMN水平并恢复运动活性的合适化合物，这些化合物可用于人体试验。最终目标是开发一种有效的药物治疗脊髓性肌萎缩症，这是婴儿死亡的主要遗传原因。 公共卫生相关性：脊髓性肌萎缩症（SMA）是肌营养不良症的一种形式，是婴儿死亡的主要遗传原因，并且以较小的破坏性形式导致儿童和成人的肌肉无力。所有形式的SMA都是由称为SMN的蛋白质水平不足引起的。SMA没有治疗方法。我们设计的策略是单独测试非常大的化合物集合，以确定其特异性增加SMN蛋白水平的能力。这项拨款申请要求资金用于表征这些化学物质的药物样性质，并在SMA小鼠模型中测试其疗效。成功将导致他们的优化，然后进入人体临床试验。

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is an autosomal recessive form of muscular dystrophy that afflicts approximately 1 in 6,000 live births and clinically manifests as progressive muscle weakness due to deterioration of motor neurons. SMA results from insufficient levels of the survival motor neuron (SMN) protein, usually due to homozygous deletion of the SMN1 gene. A nearly identical copy gene, SMN2, fails to protect from development of SMA because its mRNA undergoes alternative splicing that produces a truncated, unstable SMN protein. A small fraction of the SMN2 transcripts include exon 7 and encode the same SMN protein as SMN1. SMA severity correlates with levels of the SMN protein, with those infants with low copy number of SMN2 genes dying within two years. Children with milder forms of SMA have multiple copies of SMN2. This dose-dependency creates the opportunity for treatment of SMA by up-regulating expression of the full-length SMN protein from SMN2. We implemented a lead discovery program to identify drug-like compounds that increase SMN protein levels. Using a novel cell-based reporter assay, we performed high-throughput screens (HTS) of large chemically diverse libraries. We have validated the activities of multiple hits in secondary tests for SMN protein expression and are presently pursuing the optimization of three chemically distinct scaffolds through a focused medicinal chemistry program. Aim 1 will test the drug-like properties, pharmacokinetics and acute toxicology of active compounds that emerge from these structure-activity relationship studies. Aim 2 will examine the most active and pharmacologically suitable compounds in the "7" SMA mouse model to determine whether compound treatment increases SMN protein in vivo and whether the disease process can be mitigated. Aim 3 describes a combinatorial approach to examine the efficacy of the most promising compounds. Since compounds identified in the cell-based HTS appear to act through different mechanisms to stimulate SMN levels, a treatment approach to maximize SMN induction and minimize toxicity will be undertaken by evaluating the biological activity of pair-wise combinations of these leads. This proposal merges the efforts of three laboratories with expertise in SMA molecular biology, development of novel therapies for neurodegenerative diseases, and performance of drug trials in murine models of SMA. The predicted outcome of this proposal is identification of pharmacologically suitable compounds that increase SMN levels and restore motor activity in SMA model mice that can be advanced to human trials. The ultimate goal is to develop an effective drug treatment of spinal muscular atrophy, the leading genetic cause of infant mortality. PUBLIC HEALTH RELEVANCE: Spinal muscular atrophy (SMA), a form of muscular dystrophy, is the leading genetic cause of infant death, and in less devastating forms leads to muscle weakness in children and adults. All forms of SMA result from insufficient levels of the protein called SMN. There is no treatment for SMA. We designed a strategy is to individually test very large collections of compounds for ability to specifically increase SMN protein levels. This grant application requests funds for characterizing these chemicals' drug-like properties and testing their efficacy in a mouse model of SMA. Success would engender their optimization followed by entry into human clinical trials.