title: Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase.
abstract: Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in alpha-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl alpha-D-galactopyranoside, for a new alpha-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pK(a) of resorufin (approximately 6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease.
authors.affiliation: Imaging Probe Development Center, Division of Intramural Research, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-3708, USA.
authors.forename: Zhen-Dan
authors.initials: ZD
authors.lastname: Shi
authors.forename: Omid
authors.initials: O
authors.lastname: Motabar
authors.forename: Ehud
authors.initials: E
authors.lastname: Goldin
authors.forename: Ke
authors.initials: K
authors.lastname: Liu
authors.forename: Noel
authors.initials: N
authors.lastname: Southall
authors.forename: Ellen
authors.initials: E
authors.lastname: Sidransky
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
authors.forename: Gary L
authors.initials: GL
authors.lastname: Griffiths
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D005697:Galactosides / Q000138:chemical synthesis* / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010078:Oxazines / Q000737:chemistry; D012680:Sensitivity and Specificity; D000519:alpha-Galactosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D005697:Galactosides; D010078:Oxazines; C544135:resorufinyl alpha-D-galactopyranoside; D017485:1-Deoxynojirimycin; C014180:resorufin; C090092:migalastat; D000519:alpha-Galactosidase