title: Identification of pharmacological chaperones for Gaucher disease and characterization of their effects on beta-glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry.
abstract: Point mutations in beta-glucocerebrosidase (GCase) can result in a deficiency of both GCase activity and protein in lysosomes thereby causing Gaucher Disease (GD). Enzyme inhibitors such as isofagomine, acting as pharmacological chaperones (PCs), increase these levels by binding and stabilizing the native form of the enzyme in the endoplasmic reticulum (ER), and allow increased lysosomal transport of the enzyme. A high-throughput screen of the 50,000-compound Maybridge library identified two, non-carbohydrate-based inhibitory molecules, a 2,4-diamino-5-substituted quinazoline (IC(50) 5 microM) and a 5-substituted pyridinyl-2-furamide (IC(50) 8 microM). They raised the levels of functional GCase 1.5-2.5-fold in N370S or F213I GD fibroblasts. Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of GCase in their ER and increased levels in lysosomes. Changes in protein dynamics, monitored by hydrogen/deuterium-exchange mass spectrometry, identified a domain III active-site loop (residues 243-249) as being significantly stabilized upon binding of isofagomine or either of these two new compounds; this suggests a common mechanism for PC enhancement of intracellular transport.
authors.affiliation: Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G1X8, Canada.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Tropak
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Kornhaber
authors.forename: Brigitte A
authors.initials: BA
authors.lastname: Rigat
authors.forename: Gustavo H
authors.initials: GH
authors.lastname: Maegawa
authors.forename: Justin D
authors.initials: JD
authors.lastname: Buttner
authors.forename: Jan E
authors.initials: JE
authors.lastname: Blanchard
authors.forename: Cecilia
authors.initials: C
authors.lastname: Murphy
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Tuske
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Coales
authors.forename: Yoshitomo
authors.initials: Y
authors.lastname: Hamuro
authors.forename: Eric D
authors.initials: ED
authors.lastname: Brown
authors.forename: Don J
authors.initials: DJ
authors.lastname: Mahuran
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D041961:Deuterium Exchange Measurement; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005776:Gaucher Disease / Q000201:enzymology* / Q000473:pathology; D005962:Glucosylceramidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology; D013058:Mass Spectrometry; D009154:Mutation; D011487:Protein Conformation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D005962:Glucosylceramidase