A high-throughput screening assay using Krabbe disease patient cells

Abstract

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 as 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 showed 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 a SV40-transformed GLD patient fibroblast was measurable in high-dense microplates plates. 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 be potentially be used for the treatment of this progressive neurodegenerative disease.

Structural snapshots illustrate the catalytic cycle of β-galactocerebrosidase, the defective enzyme in Krabbe disease

“…For LSDs where the mutant enzyme possesses some residual catalytic activity, PCT is a feasible alternative treatment approach because even partial restoration of trafficking to the lysosome can provide sufficient enzyme activity to prevent disease (48). Highthroughput screening of small-molecule libraries for PCT candidates for Krabbe disease has so far been unable to identify any small molecules that significantly increase GALC activity (49). One potential PCT candidate for Krabbe disease is α-lobeline, which was shown in tissue culture to increase the activity of the hyperglycosylated mutant D528N form of GALC (50).…”

Disease specific therapies in leukodystrophies and leukoencephalopathies

“…Pharmacological chaperones, synthetic low molecular weight molecules that can be administered orally with broad body-wide distribution (including the CNS), can rescue function of mutant proteins by directing them into a proper conformation or cellular location, or protecting them from degradation [50–52]. Pharmacological chaperones that improve the activity of misfolded GALC are currently being screened, with α-lobeline and 3′,4′,7-trihydroxyisoflavone recently identified candidates [53, 54]. …”

Developing therapeutic approaches for metachromatic leukodystrophy

“…Thus, using patient-derived cell lines for HTS campaigns brings considerable advantages, compared to the traditional, single-target biochemical assays (Figure 3). 141,142 Cellular assays utilizing cells from affected patients provide a unique opportunity to assess a target protein and/or pathway in the context of potentially disrupted biochemical and/or signaling pathways secondary to the disease process. In addition, this pathogenic in cellulo setting permits the evaluation of multiple intervention points, which are potentially altered in the disease, as opposed to the commonly-used cell expression systems of a specific protein target, or a predefined step of a purified or recombinant protein-based assay (Figure 3).…”

“…In addition, this pathogenic in cellulo setting permits the evaluation of multiple intervention points, which are potentially altered in the disease, as opposed to the commonly-used cell expression systems of a specific protein target, or a predefined step of a purified or recombinant protein-based assay (Figure 3). Therefore, cell-based HTS can identify both PCs and PRs 141,142. A cell-based HTS assay was developed to identify small molecules for mutant ASAs 141.…”