Development of a HTS Assay for a Neurological Lysosomal Disease

描述（由申请人提供）：溶酶体贮积病（lsd）是由溶酶体功能必需的特定蛋白质缺陷引起的遗传性遗传病。克拉伯病，或称球状细胞白质营养不良（GLD），是一种由半乳糖脑苷酶（GALC）缺乏引起的LSD，导致包括精神碱在内的鞘糖脂积累。高水平的精神素对少突胶质细胞有毒，导致细胞凋亡和随后的脱髓鞘，这与GLD的广谱神经退行性变有关。因此，在生理上降低低浓度的精神素水平，是防止少突胶质细胞凋亡的一种有吸引力的方法，它可以转化为阻止或稳定疾病进展。造血干细胞移植（HSCT）可防止小儿GLD的暴发性神经病程，但不能防止进一步的神经恶化。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素水平的药物。在我之前筛选fda批准的化合物的工作中，我能够识别和表征两种lsd治疗剂的小分子。我的主要假设是，降低精神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具有GALC缺乏症的脑源性细胞来识别，这些细胞显示出精神素水平升高。为了验证这一假设，我计划：(I)开发一种强大的基于细胞的高通量筛选（HTS）试验，以识别能够降低GLD小鼠模型培养的脑源性细胞中升高的精神素水平的小分子；（ii）优化二级分析，包括用培养的少突胶质前体细胞进行分析，从初级筛选中选择更有效地减少精神病的小分子；（iii）对GLD小鼠模型细胞和GLD患者诱导神经元（in）细胞中的鞘脂进行综合评估，这些细胞用HTS实验选择的候选小分子处理。在HTS和二级分析的发展中，将对NCGC药理学化合物收集进行实施，其中包括几个监管机构批准的小分子。GLD的动物模型可用于测试潜在化合物，包括小鼠模型（其细胞将用于HTS试验）和狗模型。基于细胞的HTS还将识别通过间接机制减少精神病的小分子，这些小分子最终有助于更好地了解GLD的发病机制。筛选获批的药理学小分子将基于筛选化合物先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.