New Insights into the Pharmacological Chaperone Activity of C2‐Substituted Glucoimidazoles for the Treatment of Gaucher Disease

Mutations in acid β‐glucocerebrosidase (GCase) lead to the accumulation of the sphingolipid glucosylceramide, thereby resulting in Gaucher disease (GD). Active‐site‐specific competitive GCase inhibitors are effective pharmacological chaperones (PCs) that act as folding agents for mutant GCase folding in the endoplasmic reticulum. In this study, we prepared a series of glucoimidazole C2‐substituent derivatives, and evaluated their inhibition and PC properties with GCase. A cell‐based assay with patient‐derived lymphoblasts (N370S or L444P mutations) demonstrated that administration of these compounds can significantly increase GCase activity. Interestingly, the 3,3‐dimethyl‐N‐phenyl‐4‐amide‐1‐butyl‐substituted moderate inhibitor 11 had the greatest effect on activity: 2.1‐fold increase in N370S lymphoblasts at 2.5 μM and 1.2‐fold increase in L444P at 0.5 μM following a three‐day incubation. Computer docking studies and a protease protection assay were used to elucidate the ligand–enzyme interactions responsible for the chaperone activity of 11 . Western blot and immuno‐fluorescence assays verified restoration of GCase trafficking to the lysosome. Together, these results indicate that 11 is a promising PC for GD treatment and provide direct evidence of the mechanism of GCase chaperoning.