Development of a HTS Assay for a Neurological Lysosomal Disease

描述（由申请方提供）：溶酶体贮积病（LSD）是由溶酶体功能所必需的特定蛋白质缺陷引起的遗传性遗传疾病。克拉伯病或球状细胞脑白质营养不良（GLD）是一种LSD，由半乳糖苷酶（GALC）缺乏引起，导致鞘糖脂（包括精神病）积聚。高水平的精神碱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中广泛的神经变性相关。因此，降低低浓度生理学上发现的精神病碱水平是预防少突胶质细胞凋亡的有吸引力的方法，其可以转化为疾病进展的停滞或稳定。造血干细胞移植（HSCT）可以预防婴儿型GLD的暴发性神经过程，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病水平的药物。在我之前筛选FDA批准的化合物的工作中，我能够识别和表征作为两种LSD治疗剂的小分子。我的中心假设是，降低精神病肽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GALC缺乏的脑源性细胞来识别，这些细胞显示出增加的精神病水平。为了验证这一假设，我计划：（i）开发一种稳健的基于细胞的高通量筛选（HTS）试验，以鉴定降低来自GLD小鼠模型的培养脑源性细胞中升高的精神肽水平的小分子;（ii）优化二级试验，包括培养少突胶质细胞前体细胞的试验，以从初级筛选中选择更有效降低精神肽的小分子;（iii）对来自GLD小鼠模型的细胞和来自用HTS测定法选择的候选小分子治疗的GLD患者的诱导神经元（iN）细胞中的鞘脂进行综合评价。在开发本文提出的HTS和二次试验后，将根据NCGC药理学化合物集合进行实施，该集合包括几个监管机构批准的几种小分子。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试验）和犬模型。基于细胞的HTS还将鉴定通过间接机制减少精神病的小分子，这最终有助于更好地理解GLD的发病机制。根据筛选化合物之前的安全性和药代动力学记录，对获批药理学小分子的筛选将使转化为临床研究的速度更快。

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.