A high-throughput screening assay using Krabbe disease patient cells

Globoid cell leukodystrophy (GLD) or Krabbe disease is a lysosomal disease caused by β-galactocerebrosidase (GALC) deficiency resulting in a rapidly progressive neurodegenerative disorder. Unfortunately, the only available treatment is hematopoietic bone marrow transplantation, which prevents its fulminant manifestation but without treating further neurological manifestations. Here, we describe the development of a cellular high-throughput screening (HTS) assay using GLD patient fibroblasts to screen for small molecules that enhance the residual mutant GALC enzymatic activity. Small molecules have substantial therapeutic potential in GLD because they are more prone to cross the blood-brain barrier, reaching the neuronal affected cells. The transformation of primary skin fibroblasts with SV40 large T antigen has been shown to maintain the biochemical characteristics of the GLD cells and generates sufficient cells for the HTS. Using a specific fluorescent substrate, residual GALC activity from an SV40-transformed GLD patient fibroblast was measurable in high-density microplates. The pilot quantitative HTS against a small compound collection showed robust statistics. The small molecules that showed active concentration-response curves were further studied in primary GLD fibroblasts. This cell-based HTS assay demonstrates the feasibility of employing live GLD patient cells to identify therapeutic agents that can potentially be used for the treatment of this progressive neurodegenerative disease.