title: Optimization and validation of two miniaturized glucocerebrosidase enzyme assays for high throughput screening.
abstract: Glucocerebrosidase (GC) catalyzes the hydrolysis of beta-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 beta-D-glucopyranoside, have K(m) values of 768 microM and 33 microM, 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.affiliation: Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3708, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Urban
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
authors.forename: Ozlem
authors.initials: O
authors.lastname: Goker-Alpan
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.forename: Mary E
authors.initials: ME
authors.lastname: Lamarca
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.forename: Ellen
authors.initials: E
authors.lastname: Sidransky
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
mesh_terms: D004121:Dimethyl Sulfoxide; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005962:Glucosylceramidase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008904:Miniaturization; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013656:Taurocholic Acid
chemical_list: D004791:Enzyme Inhibitors; D013656:Taurocholic Acid; D005962:Glucosylceramidase; D004121:Dimethyl Sulfoxide