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

描述（由申请人提供）：Globoid-cell leukodystrophy (GLD)，通常被称为Krabbe病，是一种溶酶体贮积病（LSD），由半乳糖脑苷酶缺乏引起，半乳糖脑苷酶是一种降解含半乳糖鞘脂的溶酶体酶。当半乳糖半胱氨酸（又称精神碱）水平升高时，会对神经元细胞，尤其是少突胶质细胞造成严重的细胞毒性，导致进行性脱髓鞘。在GLD中，与其他lsd一样，除非突变导致酶的功能低于临界阈值（平均约为野生型酶的10%），否则症状不会表现出来。因此，克服这个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，从而稳定疾病过程。在大多数lsd中，相当数量的患者存在突变的溶酶体酶，但仍保留残留的酶活性。酶替代疗法（ERT）治疗几种lsd的几个方面。由于其大分子大小，ERT药物不能穿过血脑屏障（BBB），限制了对非神经症状的治疗。由于GLD的神经学表现普遍存在，如几种lsd，因此更容易穿过血脑屏障到达受影响的脑细胞的小分子药物将是非常可取的。我的中心假设是，可以在早期使用GLD患者细胞来识别帮助突变型错误折叠的GALC折叠，增强其残留酶活性的小分子

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.