Small chemical modulators of SMN biology as candidate therapeutics for SMA

描述（由申请人提供）：脊髓性肌萎缩症（SMA）是婴儿死亡最常见的遗传原因，是一种由存活运动神经元（SMN）蛋白表达减少引起的神经退行性疾病。SMA患者SMN1基因纯合缺失，并保留至少一个几乎相同的SMN2基因拷贝。SMN2基因产生低水平的SMN，导致运动神经元变性和骨骼肌萎缩。目前尚无治疗SMA的方法。因此，迫切需要确定能够恢复SMN水平或纠正SMN耗竭下游缺陷的治疗方法。由于SMN2基因的高拷贝数降低了SMA疾病的严重程度，迄今为止，大多数药物筛选工作都集中在增加SMN2基因的SMN表达上。然而，其中大多数都使用了不直接评估SMN功能的报告基因分析。因此，没有筛选作用于疾病途径而不是疾病触发因素的化合物。为了解决这些缺点，我们建立了细胞模型系统，利用哺乳动物细胞中SMN缺乏引发的增殖缺陷作为功能性SMN水平的表型读数。特别是，我们开发了一种NIH3T3细胞系，可以调节内源性小鼠SMN的敲除，其中细胞增殖依赖于人类SMN2基因产生的SMN水平。该细胞系用于建立96孔格式的细胞增殖试验，随后通过小型中试筛选验证了该试验对高通量筛选应用的适用性。在这个项目中，我们建议使用这个新开发的平台筛选一个大的化合物库，以确定SMN生物学的小分子调节剂作为候选SMA治疗药物。我们开发的系统的一个独特优势是它能够捕获通过多种作用机制起作用的化合物。在初级筛选中，hit将被定义为促进含有SMN2基因的smn缺陷NIH3T3成纤维细胞增殖的化合物。为了确认和选择活性化合物，将通过多次重复测试和专门设计的反筛试验来分析活性化合物是否具有非特异性作用。然后，确认的命中将转移到我们验证管道的下一个阶段，该阶段将采用一组正交的基于细胞的试验，旨在捕捉不同的作用机制。这将决定化合物是否增加SMN转录或剪接，增强SMN功能或影响SMN依赖的下游事件。化合物还将测试其促进从小鼠胚胎干细胞分化的SMA运动神经元存活的能力。总之，这些研究将为进一步深入分析化合物的作用机制和优先级排序提供初步的见解。我们预计这个项目将导致鉴定不同的小分子作为候选SMA治疗化合物，以推进临床前研究。这也可能导致化合物的发现，作为研究SMN生物学和疾病机制的新研究工具。

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA)-the most common genetic cause of death in infancy-is a neurodegenerative disease caused by reduced expression of the survival motor neuron (SMN) protein. SMA patients have homozygous loss of the SMN1 gene and retain at least one copy of the nearly identical SMN2 gene. The SMN2 gene produces low levels of SMN leading to motor neuron degeneration and skeletal muscle wasting. No therapy for SMA is currently available. Therefore, there is an urgent need to identify treatments that can restore SMN levels or can correct the deficits downstream of SMN depletion. Since higher copy numbers of the SMN2 gene reduce SMA disease severity, to date most drug screening efforts have focused on increasing expression of SMN from the SMN2 gene. However, most of these have used reporter assays that do not directly assess SMN function. Thus, compounds acting on the disease pathway rather than on the disease trigger are not screened for. To address these shortcomings, we established cell model systems that use proliferation defects triggered by SMN deficiency in mammalian cells as phenotypic readout of functional SMN levels. In particular, we developed a NIH3T3 cell line with regulated knockdown of endogenous mouse SMN in which cell proliferation is dependent on SMN levels produced by the human SMN2 gene. This cell line was used to establish a cell-based proliferation assay in 96-well format, and the suitability of the assay to high- throughput screening applications was subsequently validated through small pilot screens. In this project, we propose to use this newly developed platform to screen a large library of chemical compounds in order to identify small molecule modulators of SMN biology as candidate SMA therapeutics. A unique advantage of the system we developed is its capacity to capture compounds that act through multiple mechanisms of action. In the primary screen, hits will be defined as compounds that promote proliferation of SMN-deficient NIH3T3 fibroblasts that contain the SMN2 gene. For his confirmation and selection, active compounds will be analyzed by repeat testing in multiple replicates and in counter screen assays specifically designed to reveal whether they act non-specifically. Confirmed hits will then be moved to the subsequent phase in our validation pipeline, which will employ a panel of orthogonal cell-based assays designed to capture different mechanisms of action. This will determine whether compounds increase SMN transcription or splicing, enhance SMN function or influence SMN-dependent downstream events. Compounds will also be tested for their ability to promote survival of SMA motor neurons differentiated from mouse embryonic stem cells. Together, these studies will provide a first insight into mechanism(s) of action and priority ranking of compounds for future in-depth analysis. We anticipate that this project will lead to the identification of diverse small molecule as candidate SMA therapeutic compounds to advance to preclinical studies. It may also lead to the discovery of compounds as new research tools to study SMN biology and disease mechanisms.