Ambroxol and bromhexine derivatives as pharmacological chaperones for mutant glucocerebrosidase

Gaucher disease (GD) is a rare, autosomal recessive metabolic disorder of glycosphingolipid storage and is caused by mutations in the gene encoding the lysosomal hydrolase glucocerebrosidase (GCase). Certain genotypes do not lead to a complete loss-of-function, but cause a failure of the corresponding enzyme to reach a thermodynamically favoured folding state. Thus, the enzyme lacks stability, cannot pass Endoplasmic Reticulum quality control and is consequently subjected to the ER associated degradation (ERAD). Moreover, recent findings suggest that GD mutations are the most frequent genetic risk factor for the development of Parkinson symptoms, even in carrier individuals. It has recently been demonstrated that the most prevalent GD associated mutations p.N370S and p.L444P are, among others, amenable to pharmacological chaperone treatment (PCT). PCs serve as a molecular scaffolding for correct protein folding and stabilization which in turn prevents ER retention and ERAD of the enzyme. Ambroxol (ABX), an expectorant deployed in the therapy of infectious respiratory disease and an enhancer of glucocerebrosidase was described as an effective PC in GD. In the present study, we showed that ABX fully unfolds the stimulating effect in cells harboring the p.N370S mutant without inhibiting the enzyme and normalized mRNA levels of ER stress genes. Derivatives of two lead structures, ABX and the structurally related bromhexine, were able to enhance mutant GCase p.N370S and p.L444P in human fibroblasts. A further increase of enzyme activity recovery was obtained when the compounds were combined with the proteasomal inhibitor MG132. Not all compounds maintained the capability to function as a PC suggesting a distinctive structure/function relationship governing GCase activity gain. As amajor concept, ABX structures appeared more effective than bromhexine structures indicating important implications for future drug design.