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

Abstract

Ambroxol (ABX) is a mucolytic agent used for the treatment of respiratory diseases. Bioactivity has been demonstrated as an enhancement effect on lysosomal acid β-glucosidase (β-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 β-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 β-Glu, while ABX exerts its inhibitory effect in the micromolar range. In GD patient fibroblasts, IFG and ABX increase mutant β-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 β-Glu suggest different intracellular processing. In line with this observation, IFG efficiently stabilizes recombinant β-Glu against thermal denaturation in vitro, whereas ABX exerts no significant effect. Additional β-Glu enzyme activity testing using Bromohexine (BHX) and two related structures unexpectedly revealed that ABX alone can refunctionalize β-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.

Promising Effect of High Dose Ambroxol Treatment on Neurocognition and Motor Development in a Patient With Neuropathic Gaucher Disease 2

“…Furthermore, due to a link of GCase activity and alpha-Synuclein accumulation ( 27 ), analysis of this neurodegenerative marker might provide important insights into the response of the central nervous system to ambroxol. All these findings are in accordance with a recent paper by Pantoom et al who suggest that ambroxol does not solely act as a pharmacological chaperone but also has additional modes of action ( 28 ).…”

Curcumin Has Beneficial Effects on Lysosomal Alpha-Galactosidase: Potential Implications for the Cure of Fabry Disease

“…Our proposal for FD is even more promising when considering that combined therapeutic approaches for rare diseases have been proposed [ 100 , 101 , 102 , 103 , 104 ] and that most importantly, one of them was recently approved by the FDA for the treatment of cystic fibrosis [ 105 ].…”

Ambroxol as Therapy for Gaucher Disease—Ambitious but Ambivalent

“…However, because ambroxol's effects also include inhibition of pH-neutral, nonlysosomal glucocerebrosidase (GBA2), sodium channel blockade, upregulation of intracellular antioxidant and antiapoptotic signaling pathways, and antiinflammatory and immunomodulatory effects, it is uncertain to what extent reported improvements in neurological manifestations in children with GD3 are solely a function of lysosomal GCase activity enhancement. 4 Regarding ambroxol and GD1, although the safety profile seems favorable, clinical responses reported by Zhan et al 1 and Istaiti et al, 7 albeit in very different populations, appear less robust than those reported for ERTs and eliglustat. Perhaps a disconnect exists between the reported good neurological responses and the middling systemic responses, as hinted at in a recent case report 8 in which an infant with type 2 GD who started ambroxol shortly after birth had no neurological progression after 4 years yet had rapid systemic progression requiring institution of ERT.…”

“…For GD, although several potential GCase chaperone molecules have been identified by computational modeling studies over the last several years, only 1 molecule, isofagamine, an iminosugar competitive inhibitor that binds GCase in the ER and dissociates in the lysosome, has undergone clinical trial. Despite encouraging preclinical and human cell culture results showing enhanced intracellular enzyme activity, a trial of a small number of patients failed to show a meaningful clinical benefit. Migalastat for Fabry disease (α-galactosidase deficiency) is the only approved pharmacological chaperone for a lysosomal storage disease and is effective only for specific amenable variants.…”

“…On the basis of low evidentiary observational studies, high-dose oral ambroxol can decrease or reverse seizure activity in children with GD3. However, because ambroxol’s effects also include inhibition of pH-neutral, nonlysosomal glucocerebrosidase (GBA2), sodium channel blockade, upregulation of intracellular antioxidant and antiapoptotic signaling pathways, and anti-inflammatory and immunomodulatory effects, it is uncertain to what extent reported improvements in neurological manifestations in children with GD3 are solely a function of lysosomal GCase activity enhancement …”