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

描述（由申请人提供）：球样细胞脑白质营养不良（GLD），通常称为克拉伯病，是一种由半乳糖脑苷脂酶（一种降解含半乳糖鞘脂的溶酶体酶）缺乏引起的溶酶体贮积病（LSD）。在增加的水平下，半乳糖基鞘氨醇，称为精神病，对神经元细胞，特别是少突胶质细胞造成实质性的细胞毒性，导致进行性脱髓鞘。在GLD中，与其他LSD一样，除非突变导致突变酶的功能低于临界阈值（平均约为野生型酶的10%），否则不会表现出症状。因此，克服该临界阈值的缺陷溶酶体酶的增强可以防止底物积累和细胞功能障碍，从而导致疾病过程的稳定。在大多数LSD中，大量患者存在突变型溶酶体酶，其仍保留残余酶活性。酶替代疗法（ERT）治疗一些LSD的几个方面。由于它们的大分子尺寸，ERT药剂不能穿过血脑屏障（BBB），限制了对非神经症状的治疗。由于神经学表现在GLD中占优势，如在几种LSD中一样，更倾向于穿过BBB到达受影响的脑细胞的小分子药剂将是高度期望的。我的中心假设是，可以在早期阶段使用GLD患者细胞鉴定出有助于错误折叠的突变GALC折叠的小分子，增强其残余酶活性 高通量筛选（HTS）由于计划的HTS试验是一项细胞试验，因此将选择通过与GALC直接和间接相互作用增强残留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。测试我的中心假设的目的是：（i）使用GLD患者细胞系实施定量（q）HTS测定，以鉴定能够产生突变GALC增强的小分子;（ii）验证通过基于测试对GALC的作用的特异性的测定、精神碱还原测定和易位测定的二级测定鉴定的小分子;（iii）通过检测ER蛋白折叠和降解途径中涉及的元件，进行三级测定以评估一些小分子促进突变型GALC增强的机制。针对分子文库生产中心网络实施基于细胞的HTS测定将导致鉴定小分子作为用于辅助GALC错误折叠突变体的折叠的候选“命中物”。这些小分子将是患有GLD的患者的潜在治疗剂。在基于细胞的qHTS测定中，一些筛选的化合物不一定与突变体GALC直接相互作用，但影响调节蛋白质折叠和降解的替代途径。因此，这些小分子可能具有更广泛的应用，作为GLD和其他LSD的潜在疗法，并最终作为其他构象错误蛋白质疾病的潜在疗法。

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.