title: Cell-based high-throughput screening identifies galactocerebrosidase enhancers as potential small-molecule therapies for Krabbe's disease.
abstract: Krabbe's disease, also known as globoid cell leukodystrophy (GLD), is a lysosomal storage disease caused by the deficiency of the lysosomal enzyme β-galactocerebrosidase (GALC), resulting in severe neurological manifestations related to demyelination secondary to elevated galactosylsphingosine (psychosine) with its subsequent cytotoxicity. The only available treatment is hematopoietic stem cell transplantation, which delays disease onset but does not prevent long-term neurological manifestations. This article describes the identification of small molecules that enhance mutant GALC activity, identified by quantitative cell-based high-throughput screening (qHTS). Using a specific neurologically relevant murine cell line (145M-Twi) modified to express common human hGALC-G270D mutant, we were able to detect GALC activity in a 1,536-well microplate format. The qHTS of approximately 46,000 compounds identified three small molecules that showed significant enhancements of residual mutant GALC activity in primary cell lines from GLD patients. These compounds were shown to increase the levels of GALC-G270D mutant in the lysosomal compartment. In kinetic assessments, these small molecules failed to disturb the GALC kinetic profile under acidic conditions, which is highly desirable for folding-assisting molecules operating in the endoplasmic reticulum and not affecting GALC catalytic properties in the lysosomal compartment. In addition, these small molecules rescued the decreased GALC activity at neutral pH and partially stabilized GALC under heat-denaturating conditions. These drug-like compounds can be used as the starting point to develop novel small-molecule agents to treat the progressive neurodegenerative course of GLD. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Pediatrics, University of Florida, Gainesville, Florida.
authors.forename: Dae Song
authors.initials: DS
authors.lastname: Jang
authors.affiliation: National Institutes of Health Center for Advancing Translational Sciences, Rockville, Maryland.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Ye
authors.affiliation: Department of Pediatrics, University of Florida, Gainesville, Florida.
authors.forename: Tian
authors.initials: T
authors.lastname: Guimei
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, Maryland.
authors.forename: Melani
authors.initials: M
authors.lastname: Solomon
authors.affiliation: National Institutes of Health Center for Advancing Translational Sciences, Rockville, Maryland.
authors.forename: Noel
authors.initials: N
authors.lastname: Southall
authors.affiliation: National Institutes of Health Center for Advancing Translational Sciences, Rockville, Maryland.
authors.forename: Xin
authors.initials: X
authors.lastname: Hu
authors.affiliation: National Institutes of Health Center for Advancing Translational Sciences, Rockville, Maryland.
authors.forename: Juan
authors.initials: J
authors.lastname: Marugan
authors.affiliation: National Institutes of Health Center for Advancing Translational Sciences, Rockville, Maryland.
authors.forename: Marc
authors.initials: M
authors.lastname: Ferrer
authors.affiliation: Department of Pediatrics, University of Florida, Gainesville, Florida. gmaegawa@ufl.edu.|Department of Neuroscience, University of Florida, Gainesville, Florida. gmaegawa@ufl.edu.|Department of Microbiology and Immunology, University of Florida, Gainesville, Florida. gmaegawa@ufl.edu.
authors.forename: Gustavo H B
authors.initials: GH
authors.lastname: Maegawa
mesh_terms: D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000201:enzymology; D005698:Galactosylceramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007965:Leukodystrophy, Globoid Cell / Q000188:drug therapy* / Q000473:pathology; D009154:Mutation / Q000235:genetics; D011107:Polylysine / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry*; D014162:Transfection
chemical_list: D054852:Small Molecule Libraries; D011107:Polylysine; D005698:Galactosylceramidase
keywords: Krabbe's disease; quantitative high-throughput screening; small molecules; β-galactocerebrosidase