Journal
COMBINATORIAL CHEMISTRY & HIGH THROUGHPUT SCREENING
Volume 11, Issue 10, Pages 817-824Publisher
BENTHAM SCIENCE PUBL LTD
DOI: 10.2174/138620708786734244
Keywords
Glucocerebrosidase; beta-glucosidase; Gaucher disease; small molecule; assay optimization; assay miniaturization; HTS; quantitative high throughput screening; qHTS
Funding
- NIH
- National Human Genome Research Institute
Ask authors/readers for more resources
Glucocerebrosidase (GC) catalyzes the hydrolysis of glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene (GBA) 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 inhibitors of GC have been shown to act as chemical chaperones, stabilizing the conformation of mutant proteins and thus restoring their function. High throughput screening (HTS) of small molecule libraries for such compounds with potential for chaperone therapy requires an accurate, reproducible and sensitive assay method. We have adapted and optimized two fluorogenic GC enzyme assays and miniaturized them into the 1536-well plate format for HTS. The two substrates, 4-methylumbelliferyl beta-D-glucopyranoside and resorufin D-glucopyranoside, have K-m values of 768 mu M and 33 mu M, respectively, and different emission spectra. Paired screening with the two assays helps to eliminate false inference of activity due to autofluorescence or fluorescence quenching by the screened compounds. Test screens with the LOPAC library indicated that both assays were robust for HTS, and gave comparable results for GC inhibitor activities. These two assays can be used to identify both GC activators and inhibitors with potential therapeutic value.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available