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Identification of Pharmacological Chaperones for Gaucher Disease and Characterization of Their Effects on β‐Glucocerebrosidase by Hydrogen/Deuterium Exchange Mass Spectrometry
Author(s) -
Tropak Michael B.,
Kornhaber Gregory J.,
Rigat Brigitte A.,
Maegawa Gustavo H.,
Buttner Justin D.,
Blanchard Jan E.,
Murphy Cecilia,
Tuske Steven J.,
Coales Stephen J.,
Hamuro Yoshitomo,
Brown Eric D.,
Mahuran Don J.
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200800304
Subject(s) - glucocerebrosidase , chemistry , endoplasmic reticulum , hydrogen–deuterium exchange , enzyme , biochemistry , intracellular , mass spectrometry , chromatography
Point mutations in β‐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 μ M ) and a 5‐substituted pyridinyl‐2‐furamide (IC 50 8 μ M ). 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.