Development of a HTS Assay for a Neurological Lysosomal Disease

描述(由申请人提供)：溶酶体储存疾病(LSD)是一种遗传性遗传疾病，由溶酶体功能所必需的特定蛋白质的缺陷引起。Krabbe病，或球状细胞白质营养不良(GLD)，是一种由于半乳糖脑苷酶(GALC)缺乏导致包括神经氨酸在内的神经鞘糖脂积聚而引起的LSD。高水平的心磷脂对少突胶质细胞有毒性，导致细胞凋亡和随后的脱髓鞘，这与GLD广泛的神经变性有关。因此，降低低浓度的神经氨酸水平是防止少突胶质细胞凋亡的一个有吸引力的方法，这可以转化为阻止或稳定疾病进展。造血干细胞移植(HSCT)阻止了婴儿形式的GLD的暴发性神经过程，但未能阻止进一步的神经退化。小分子更有可能穿过血脑屏障，因此可以用作降低大脑中精神药物水平的药物。在我之前的工作中，我筛选了FDA批准的化合物，我能够识别和表征小分子作为两种LSD的治疗剂。我的中心假设是，降低精神病素水平的特定小分子将对GLD具有显著的治疗潜力，以防止少突胶质细胞凋亡，并最终控制或阻止神经症状的进展。这些小分子最有可能通过培养的患有GALC缺乏症的脑源性细胞来鉴定，GALC缺乏症显示出精神碱水平的增加。为了验证这一假设，我计划：(I)建立一种可靠的基于细胞的高通量筛选(HTS)分析方法，以确定能降低GLD小鼠模型小鼠脑源性细胞中升高的精神碱水平的小分子；(Ii)优化二次分析，包括对培养的少突胶质细胞前体细胞进行分析，以从初次筛选中选择更有效地降低精神肽水平的小分子；(Iii)对GLD小鼠模型细胞以及经HTS分析选择的候选小分子治疗的GLD患者的神经鞘糖脂进行全面评估。根据HTS和这里提出的二次检测的发展，将针对NCGC药理化合物集合进行实施，其中包括几个监管机构批准的几个小分子。GLD的动物模型可用于测试潜在的化合物，包括小鼠(其细胞将用于HTS测试)和狗模型。基于细胞的HTS还将通过间接机制识别降低精神药物的小分子，最终合作更好地了解GLD的发病机制。对已批准的药理小分子进行筛选将使根据筛选出的化合物以前的安全性和药代动力学记录更快地转化为临床研究。

DESCRIPTION (provided by applicant): Lysosomal storage diseases (LSDs) are inherited genetic conditions caused by the defect in a specific protein that is essential for lysosomal function. Krabbe disease, or globoid-cell leukodystrophy (GLD), is a LSD caused by ¿-galactocerebrosidase (GALC) deficiency, resulting in accumulation of glycosphingolipids including psychosine. High levels of psychosine are toxic to oligodendrocytes, resulting in apoptosis and subsequent demyelination, which correlates to the wide spectrum of neurodegeneration in GLD. Therefore, reducing the levels of psychosine, found physiologically at low concentrations, is an attractive approach to prevent oligodendrocyte apoptosis, which can translate in arrest or stabilization of disease progression. Hematopoietic stem cell transplantation (HSCT) prevents the fulminant neurological course of the infantile form of GLD, but fails to prevent further neurodeterioration. Small molecules are more likely to cross the blood-brain barrier and consequently can be used as agents to reduce psychosine levels in the brain. In my previous work screening FDA-approved compounds, I was able to identify and characterize small molecules as therapeutic agents for two LSDs. My central hypothesis is that specific small molecules that reduce the levels of psychosine will have significant therapeutic potential for GLD to prevent oligodentrocyte apoptosis and ultimately controlling or arresting the progression of neurological symptoms. These small molecules are most likely to be identified through cultured brain-derived cells with GALC deficiency, which show increased levels of psychosine. To test this hypothesis, I plan to: (i) develop of a robust cell-based high-throughput screening (HTS) assay to identify small molecules that reduce the elevated psychosine levels in cultured brain-derived cells from mouse model of GLD; (ii) optimize secondary assays including assays with cultured oligodendrocyte precursor cells to select small molecules from the primary screening that more effectively reduce the psychosine; (iii) perform a comprehensive evaluation of sphingolipids in cells from GLD mouse model and in induced-neuronal (iN) cells from GLD patients treated with candidate small molecules selected by HTS assay. Upon the development of the HTS and secondary assays here proposed, the implementation will be done against the NCGC Pharmacological Compound Collection, which includes several small molecules approved by several regulatory bodies. Animal models of GLD are available for testing the potential compounds including the mouse (whose cells will be used in HTS assay) and dog models. Cell-based HTS will also identify small molecules that reduce psychosine by indirect mechanisms, which ultimately collaborate to a better understanding on the pathogenesis of GLD. The screening of approved pharmacological small molecules will make the translation to clinical studies faster based on previous safety and pharmacokinetics records of the screened compounds.