A Novel Cell-Based Assay to Identify Small Molecules for B -Galactocerebrosidase

描述(申请人提供)：球状细胞性脑白质营养不良(GLD)，通常被称为Krabbe病，是一种溶酶体储存性疾病(LSD)，由β-半乳糖脑苷酶缺乏引起，-Galatocerebrosidase是一种溶酶体酶，可降解含半乳糖的鞘糖脂。当水平增加时，半乳糖鞘氨醇，也就是神经氨酸，对神经细胞，特别是少突胶质细胞造成大量的细胞毒性，导致进行性脱髓鞘。在GLD中，就像在其他LSD中一样，除非突变导致突变酶的功能低于临界阈值，即平均~10%的野生型酶，否则不会出现症状。因此，加强缺乏溶酶体的酶，以克服这一关键阈值，可以防止底物积累和细胞功能障碍，导致疾病过程的稳定。在大多数LSD中，相当数量的患者存在突变的溶酶体酶，但仍保持残留酶活性。酶替代疗法(ERT)治疗一些LSD的几个方面。由于分子尺寸大，ERT药物无法通过血脑屏障(BBB)，将治疗限制在非神经症状。由于神经学表现在GLD中占主导地位，就像在几个LSD中一样，小分子药物更容易通过血脑屏障达到受影响的脑细胞，将是非常可取的。我的中心假设是，帮助突变的错误折叠的GALC折叠，增强其残留酶活性的小分子，可以在GLD患者细胞的早期阶段被识别出来。 高通量筛选(HTS)。由于计划中的HTS检测是一种细胞检测，将选择通过与GALC直接和间接相互作用来增强残留GALC活性的两种小分子。该提案的最终目标是发现可用于治疗GLD患者的小分子。我的中心假设的目的是：(1)利用GLD患者细胞系实施(Q)HTS定量检测，以确定能够产生突变GALC增强的小分子；(2)基于测试对GALC的影响的特异性的检测、精神苷还原检测和易位检测的二次检测确定的小分子；(3)进行第三级检测以通过检测ER蛋白折叠和降解途径中涉及的元件来评估一些小分子促进突变GALC增强的机制。针对分子文库生产中心网络的基于细胞的HTS测试的实施将导致将小分子确定为辅助GALC错误折叠突变体的候选“HITS”。这些小分子将成为治疗GLD患者的潜在药物。在基于细胞的qHTS分析中，一些筛选的化合物不一定会与突变的GALC直接相互作用，但会影响调节蛋白质折叠和降解的替代途径。因此，这些小分子可能会有更广泛的应用，作为GLD和其他LSD的潜在疗法，并最终治疗其他构象错误的蛋白质疾病。

DESCRIPTION (provided by applicant): Globoid-cell leukodystrophy (GLD), mostly known as Krabbe disease, is a lysosomal storage disease (LSD) caused by the deficiency of ¿-galatocerebrosidase, a lysosomal enzyme that degrades galactose-containing sphingolipids. At increased levels, galactosylsphingosine, known as psychosine, causes substantial cytotoxicity to neuronal cells, especially oligodendrocytes resulting in progressive demyelination. In GLD, as in other LSDs, symptoms are not manifested unless mutations result into a mutant enzyme that functions below a critical threshold, of on average ~10% of wild type enzyme. Therefore, enhancements of the deficient lysosomal enzyme that overcome this critical threshold can prevent substrate accumulation and cellular dysfunction, resulting in stabilization of the disease process. In most LSDs, substantial number of patients present with mutant lysosomal enzymes that still retains a residual enzymatic activity. The enzyme replacement therapy (ERT) treats several aspects of a few LSDs. Due to their large molecular size, ERT agents are unable to cross the blood-brain barrier (BBB), restricting treatment to non-neurological symptoms. Since the neurological manifestations prevail in GLD, as in several LSDs, small molecules agents, which are more prone to cross the BBB achieving affected brain cells, will be highly desirable. My central hypothesis is that small molecules that assist the folding of mutant misfolded GALC, enhancing its residual enzymatic activity, can be identified using GLD patient cells at early stage of the high throughput screening (HTS). Since the planned HTS assay is a cellular assay, both small molecules that enhance residual GALC activity by direct and indirect interactions with GALC will be selected. The final goal of the proposal is discover small molecules that can be used as therapies for patients suffering from GLD. The aims to test my central hypothesis are: (i) to implement a quantitative (q)HTS assay using GLD patient cell lines to identify small molecules that are able to produce enhancements of mutant GALC; (ii) to validate the small molecules identified by secondary assays based on assays to test specificity of effects on GALC, psychosine reduction assays and translocation assays; (iii) to perform tertiary assays to evaluate the mechanisms some small molecules promote mutant GALC enhancement by examining elements involved in ER protein folding and degradation pathways. The implementation of the cell-based HTS assay against the Molecular Libraries Production Centers Network will result in the identification of small molecules as candidate "hits" for assisting foldng of GALC misfolded mutants. These small molecules will be potential therapeutic agents to patients suffering from GLD. In cell-based qHTS assay, some of the screened compounds will not necessarily directly interact with mutant GALC, but affect alternative pathways that regulate protein folding and degradation. Consequently, these small molecules may have a more broad application as potential therapies for GLD and other LSDs, and eventually other misconformation protein diseases.