title: High-throughput screening for human lysosomal beta-N-Acetyl hexosaminidase inhibitors acting as pharmacological chaperones.
abstract: The adult forms of Tay-Sachs and Sandhoff diseases result when the activity of beta-hexosaminidase A (Hex) falls below approximately 10% of normal due to decreased transport of the destabilized mutant enzyme to the lysosome. Carbohydrate-based competitive inhibitors of Hex act as pharmacological chaperones (PC) in patient cells, facilitating exit of the enzyme from the endoplasmic reticulum, thereby increasing the mutant Hex protein and activity levels in the lysosome 3- to 6-fold. To identify drug-like PC candidates, we developed a fluorescence-based real-time enzyme assay and screened the Maybridge library of 50,000 compounds for inhibitors of purified Hex. Three structurally distinct micromolar competitive inhibitors, a bisnaphthalimide, nitro-indan-1-one, and pyrrolo[3,4-d]pyridazin-1-one were identified that specifically increased lysosomal Hex protein and activity levels in patient fibroblasts. These results validate screening for inhibitory compounds as an approach to identifying PCs.
authors.affiliation: Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Tropak
authors.forename: Jan E
authors.initials: JE
authors.lastname: Blanchard
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
authors.forename: Eric D
authors.initials: ED
authors.lastname: Brown
authors.forename: Don
authors.initials: D
authors.lastname: Mahuran
mesh_terms: D000328:Adult; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005347:Fibroblasts; D006801:Humans; D007189:Indans / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008247:Lysosomes / Q000201:enzymology*; D018832:Molecular Chaperones / Q000494:pharmacology*; D053644:Naphthalimides / Q000494:pharmacology; D011724:Pyridazines / Q000494:pharmacology; D012497:Sandhoff Disease / Q000188:drug therapy / Q000201:enzymology*; D013661:Tay-Sachs Disease / Q000188:drug therapy / Q000201:enzymology*; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007189:Indans; D018832:Molecular Chaperones; D053644:Naphthalimides; D011724:Pyridazines; D001619:beta-N-Acetylhexosaminidases