Identification of Pharmacological Chaperones for Gaucher Disease and Characterization of Their Effects on β-Glucocerebrosidase by Hydrogen/Deuterium Exchange Mass Spectrometry

Point mutations in beta-glucocerebrosidase (GCase) can result in a deficiency of both GCase activity and protein in lyssosomes 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 50000-compound Maybridge library identified two, non-carbohydrate-based inhibitory molecules, a 2,4-diamino-5-substituted quinazoline (IC50 5 mu M) and a 5-substituted pyridinyl-2-furamide (IC50 8 mu M). They raised the levels of functional GCase 1.5-2.5-fold in N370S or F2131 GD fibrolasts Immunofluorescence confirmed that treated GD fibroblasts had decreased levels of GCase of 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.