The iminosugar isofagomine increases the activity of N370S mutant acid β-glucosidase in Gaucher fibroblasts by several mechanisms

Abstract

Gaucher disease is a lysosomal storage disorder caused by deficiency in lysosomal acid ␤-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. One of the most prevalent disease-causing mutations, N370S, results in an enzyme with lower catalytic activity and impaired exit from the endoplasmic reticulum. Here, we report that the iminosugar isofagomine (IFG), an active-site inhibitor, increases GlcCerase activity 3.0 ؎ 0.6-fold in N370S fibroblasts by several mechanisms. A major effect of IFG is to facilitate the folding and transport of newly synthesized GlcCerase in the endoplasmic reticulum, thereby increasing the lysosomal pool of the enzyme. In addition, N370S GlcCerase synthesized in the presence of IFG exhibits a shift in pH optimum from 6.4 to 5.2 and altered sensitivity to SDS. Although IFG fully inhibits GlcCerase in the lysosome in an in situ assay, washout of the drug leads to partial recovery of GlcCerase activity within 4 h and full recovery by 24 h. These findings provide support for the possible use of active-site inhibitors in the treatment of some forms of Gaucher disease.

Isofagomine Induced Stabilization of Glucocerebrosidase

“…Currently, isofagomine (IFG), an iminosugar and potent competitive inhibitor of GCase (Plicera, Amicus Therapeutics Inc.) is being evaluated clinically as a PC for EET. [6][7][8] IFG, like other GCase PCs, is a competitive inhibitor with IC 50 values of 5 and 30 nM at pH 7.2 and pH 5.2, respectively. [8] Surprisingly, it has been shown to be effective as a PC in cultured patient cells only at concentrations significantly higher than its IC 50 , 10-100 μM.…”

“…[6][7][8] IFG, like other GCase PCs, is a competitive inhibitor with IC 50 values of 5 and 30 nM at pH 7.2 and pH 5.2, respectively. [8] Surprisingly, it has been shown to be effective as a PC in cultured patient cells only at concentrations significantly higher than its IC 50 , 10-100 μM. [8] This likely reflects the fact that the efficacy of a PC not only depends on its affinity for the target enzyme but also on its bioavailability in terms of membrane permeability, subcellular distribution and metabolism.…”

“…[8] Surprisingly, it has been shown to be effective as a PC in cultured patient cells only at concentrations significantly higher than its IC 50 , 10-100 μM. [8] This likely reflects the fact that the efficacy of a PC not only depends on its affinity for the target enzyme but also on its bioavailability in terms of membrane permeability, subcellular distribution and metabolism. [9] Thus, the actual intracellular concentration of IFG after treatment is likely much less than the extracellular concentration.…”

“…[14,15] Cell-based activity and immunofluorescence assays have demonstrated an IFGdependent increase in lysosomal pools of enzymatically active N370S GCase. [7,8,16] Steet et al [8] proposed that IFG increases the exit of mutant GCase from the ER. Given that the kinetic properties of the mutant enzyme following treatment with IFG resembled those of the wild type enzyme, it was further proposed that the increased ER export occurred because of the stabilization of natively folded N370S GCase, which was induced by the formation of IFG-GCase complexes.…”

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…”