Chemical chaperones increase the cellular activity of N370S β-glucosidase: A therapeutic strategy for Gaucher disease

Abstract

Gaucher disease is a lysosomal storage disorder caused by deficient lysosomal ␤-glucosidase (␤-Glu) activity. A marked decrease in enzyme activity results in progressive accumulation of the substrate (glucosylceramide) in macrophages, leading to hepatosplenomegaly, anemia, skeletal lesions, and sometimes CNS involvement. Enzyme replacement therapy for Gaucher disease is costly and relatively ineffective for CNS involvement. Chemical chaperones have been shown to stabilize various proteins against misfolding, increasing proper trafficking from the endoplasmic reticulum. We report herein that the addition of subinhibitory concentrations (10 M) of N-(n-nonyl)deoxynojirimycin (NN-DNJ) to a fibroblast culture medium for 9 days leads to a 2-fold increase in the activity of N370S ␤-Glu, the most common mutation causing Gaucher disease. Moreover, the increased activity persists for at least 6 days after the withdrawal of the putative chaperone. The NN-DNJ chaperone also increases WT ␤-Glu activity, but not that of L444P, a less prevalent Gaucher disease variant. Incubation of isolated soluble WT enzyme with NN-DNJ reveals that ␤-Glu is stabilized against heat denaturation in a dose-dependent fashion. We propose that NN-DNJ chaperones ␤-Glu folding at neutral pH, thus allowing the stabilized enzyme to transit from the endoplasmic reticulum to the Golgi, enabling proper trafficking to the lysosome. Clinical data suggest that a modest increase in ␤-Glu activity may be sufficient to achieve a therapeutic effect.

Selective action of the iminosugar isofagomine, a pharmacological chaperone for mutant forms of acid-β-glucosidase

“…While both treatments are effective in improving the hematologic and visceral symptoms in patients with mild, non-neuronopathic forms of the disease, their efficacy may be limited by their inability to cross the blood-brain barrier (ERT) or adverse side effects (SRT). Recent studies have demonstrated the ability of sugar analog inhibitors to increase the activity of mutant lysosomal enzymes in patient fibroblasts by acting as pharmacological chaperones [10][11][12][13][14][15]. As active site inhibitors, these compounds bind to the mutant enzymes and stabilize them in the ER, thereby preventing their ER-associated degradation and facilitating their folding and transport out of this compartment to lysosomes.…”

“…Since fibroblasts derived from different N370S/N370S Gaucher patients can exhibit variable GlcCerase residual activity [2,10,15], we tested the ability of IFG to enhance enzyme activity from multiple N370S patient fibroblasts. The results are summarized in Table I.…”

“…Several researchers have demonstrated the ability of iminosugars to increase the activity of mutant forms of GlcCerase [10][11][12][13][14][15]. We have recently reported that the iminosugar, isofagomine (IFG), enhances N370S GlcCerase activity by approximately 3-fold by facilitating its folding and transport out of the ER to lysosomes.…”

Isofagomine Induced Stabilization of Glucocerebrosidase

“…[7] Two GD patient fibroblast cell lines, one harboring N370S/N370S and the other F213I/L444P GCase mutations, were treated with IFG for five days and intracellular GCase activity following lysis of cells was determined with the aid of the artificial substrate 4-methylum-belliferylbeta-D-glucopyranoside (MUGlc). [30,31] An increase in enzymatic activity is observed as an increase in fluorescence upon release of the 4-methylumbelliferyl fluorophore. Treatment of N370S/N370S and F213I/L444P fibroblasts with 25 μM IFG for five days enhanced GCase activity 2.5-and 4.3-fold over non-pretreated cells, respectively (see Figure 1C).…”

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…”