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.
The implementation of the cell-based high throughput screening assay in cells from patients with globoid-cell leukodystrophy (GLD) patients, mostly known as Krabbe disease, will select therapeutic small molecules that assist the folding of misfolded ?-galactocerebrosidase GALC mutants, enhancing their final residual enzymatic activity. Once these selected GALC enhancers are characterized and validated, potential drug(s) to treat this devastating neurological lysosomal storage disorder will be identified.
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