Mechanistic Insight into the Mode of Action of Acid beta-Glucosidase Enhancer Ambroxol

Ambroxol (ABX) is a mucolytic agent used for the treatment of respiratory diseases. Bioactivity has been demonstrated as an enhancement effect on lysosomal acid beta-glucosidase (beta-Glu) activity in Gaucher disease (GD). The positive effects observed have been attributed to a mechanism of action similar to pharmacological chaperones (PCs), but an exact mechanistic description is still pending. The current study uses cell culture and in vitro assays to study the effects of ABX on beta-Glu activity, processing, and stability upon ligand binding. Structural analogues bromohexine, 4-hydroxybromohexine, and norbromohexine were screened for chaperone efficacy, and in silico docking was performed. The sugar mimetic isofagomine (IFG) strongly inhibits beta-Glu, while ABX exerts its inhibitory effect in the micromolar range. In GD patient fibroblasts, IFG and ABX increase mutant beta-Glu activity to identical levels. However, the characteristics of the banding patterns of Endoglycosidase-H (Endo-H)-digested enzyme and a substantially lower half-life of ABX-treated beta-Glu suggest different intracellular processing. In line with this observation, IFG efficiently stabilizes recombinant beta-Glu against thermal denaturation in vitro, whereas ABX exerts no significant effect. Additional beta-Glu enzyme activity testing using Bromohexine (BHX) and two related structures unexpectedly revealed that ABX alone can refunctionalize beta-Glu in cellula. Taken together, our data indicate that ABX has little in vitro ability to act as PC, so the mode of action requires further clarification.