TY - JOUR
T1 - Cell-based high-throughput screening identifies galactocerebrosidase enhancers as potential small-molecule therapies for Krabbe's disease
AU - Jang, Dae Song
AU - Ye, Wenjuan
AU - Guimei, Tian
AU - Solomon, Melani
AU - Southall, Noel
AU - Hu, Xin
AU - Marugan, Juan
AU - Ferrer, Marc
AU - Maegawa, Gustavo H.B.
N1 - Funding Information:
The authors thank colleagues at Johns Hopkins University and University of Florida who indirectly assisted in the experiments described here; CiventiChem Inc., from which the HMU GALC-specific substrate was purchased; G. Gaslini Institute-Telethon Genetic Biobank Network (project No. GTB07001) for one of the cell lines used from patients affected by Krabbe's disease; and Mirella Filocamo, PhD, Head of Laboratory Diagnosi Pre- e Postnatale and Malattie Metaboliche and Coordinator Telethon Genetic Biobank Network and Gaslini Institute, Genova, Italy.
Publisher Copyright:
© 2016 Wiley Periodicals, Inc.
PY - 2016/11/1
Y1 - 2016/11/1
N2 - 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.
AB - 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.
KW - Krabbe's disease
KW - quantitative high-throughput screening
KW - small molecules
KW - β-galactocerebrosidase
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U2 - 10.1002/jnr.23875
DO - 10.1002/jnr.23875
M3 - Article
C2 - 27638606
AN - SCOPUS:84990206918
SN - 0360-4012
VL - 94
SP - 1231
EP - 1245
JO - Journal of neuroscience research
JF - Journal of neuroscience research
IS - 11
ER -