Identification and Characterization of Ambroxol as an Enzyme Enhancement Agent for Gaucher Disease

Abstract

Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243-249, 310 -312, and 386 -400 near the active site of GCase are stabilized. Consistent with its mixedtype inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes.

Glucocerebrosidase mutations and the pathogenesis of Parkinson disease

“…ABX, a mixed-type inhibitor of GCase (Maegawa et al, 2009), has been shown to interact with both the active and non-active sites of GCase in a pHdependent manner. It has a theoretical advantage over other iminosugars because it does not require wash-out periods, which enables continued dosing.…”

“…In an experiment to examine the "chaperoning" half-life of ABX, GD fibroblasts were grown in medium containing 60μm ABX or DMSO (mock-treated) for 5 days (Maegawa et al, 2009). The cells were then "chased" for 8 days by replacing the ABX/DMSO-containing media with fresh media lacking the compounds and thereafter every other day.…”

Ambroxol effects in glucocerebrosidase and α‐synuclein transgenic mice

“…To this end, small molecular chaperones designed to cross the blood–brain barrier that are capable of increasing GCase activity are being investigated as a novel therapy for PD to decrease α‐synuclein levels 17, 18, 19, 20, 21, 22, 23, 24, 25…”

“…Ambroxol was identified as a GCase chaperone after screening the library of US Food and Drug Administration–approved drugs with a thermal denaturation assay using wild‐type GCase 19. To date, 2 ambroxol studies using wild‐type mice or transgenic mice carrying a human transgene containing either N370S or L444P mutation failed to provide convincing evidence to determine whether ambroxol is capable of increasing GCase in the peripheral and neuronal tissues 23, 24.…”

Evaluation of N-nonyl-deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy

“…However, the ideal PC would bind tightest at the neutral pH of the ER and weakest or not at all at the acidic pH of the lysosome [11], e.g. Ambroxol for Gaucher Disease [12]. Like SRT, EET has the potential to treat the CNS, but is limited to a subgroup of "responsive" mutations.…”