qHTS for Activators of Human Glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Fibroblast Translocation
Glucocerebrosidase (GCase) catalyzes the hydrolysis of beta-glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene result in Gaucher disease, an autosomal recessive lysosomal storage disorder. Many of the mutations encountered in patients with Gaucher disease are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of more ..
BioActive Compounds: 2
Glucocerebrosidase (GCase) catalyzes the hydrolysis of beta-glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene result in Gaucher disease, an autosomal recessive lysosomal storage disorder. Many of the mutations encountered in patients with Gaucher disease are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of this lysosomal protein. Some GCase inhibitors have been shown to act as chemical chaperones, stabilizing the conformation of mutant proteins and thus restoring their function. However, the enhancement of enzyme activity by the chaperone action of an enzyme inhibitor must be balanced against the direct inhibition of the enzyme. An enzyme activator could also function as a chaperone by binding to the enzyme and helping to correct its misfolding and mistrafficking. Activators for GCase have not yet been identified, and they may have better therapeutic potential than inhibitors. Therefore the discovery and development of chemical activators may provide a new strategy for the chaperone therapy.
The probe that has been identified was tested in a chaperone translocation experiment in human fibroblasts. This assay attempts to quantitate translocated glucocerebrosidase protein in patient-derived fibroblasts following extended compound incubation. The fibroblasts tested in this experiment were homozygous either for N370S glucocerebrosidase or wildtype GCase.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
MLPCN Grant: MH086442-01
Assay Submitter (PI): Wei Zheng
Primary dermal fibroblasts derived from skin biopsies from two previously described N370S/N370S Gaucher patients and a control were seeded in Lab-Tek 4 chamber slides (Fisher Scientific, Pittsburgh, PA). After compound treatment, fibroblasts were fixed in 3% paraformaldehyde. The cells were permeabelized with 0.1 % Triton-X for 10 min. and blocked in PBS containing 0.1% saponin, 100 microM glycine, 0.1% BSA and 2% donkey serum. This is followed by an incubation with mouse monoclonal anti-LAMP1 or LAMP-2 (1:100, Developmental Studies Hybridoma bank, University of Iowa, Iowa City, IA) and the rabbit polyclonal anti-GCase R386 antibody (1:500); the cells were washed and incubated with secondary donkey anti-mouse or anti-rabbit antibodies conjugated to ALEXA-488 or ALEXA-555, respectively (Invitrogen, Carlsbad, CA), washed again, and mounted in VectaShield with DAPI (Vector Laboratories, Burlingame, CA).
Cells were imaged with a Zeiss 510 META confocal laser-scanning microscope (Carl Zeiss, Microimaging Inc., Germany) using an Argon (458, 477, 488, 514 nm) 30 mW laser, a HeNe (543 nm) 1 mW laser, and a laser diode (405 nm). Low and high magnification images were acquired using a Plan-Apochromat 20X/0.75 objective and a Plan-Apochromat 100x/1.4 oil DIC objective, respectively. Images were taken with the same laser settings and all the images shown are collapsed z-stacks.
This is a translocation in human fibroblast measuring immunostaining as observed through a microscope. If translocation was visually observed, the compounds are considered "active"; if no translocation was observed, compounds are considered "inactive"; if the results is unclear, compounds are considered "inconclusive".
Active compounds are assigned a score of 50, inactive compounds are assigned a score of 0, inconclusive compounds are assigned a score of 10.
Data Table (Concise)