Hydrophilic iminosugar active‐site‐specific chaperones increase residual glucocerebrosidase activity in fibroblasts from Gaucher patients

Abstract

Gaucher disease is an autosomal recessive lysosomal storage disorder caused by the deficient activity of glucocerebrosidase. Accumulation of glucosylceramide, primarily in the lysosomes of cells of the reticuloendothelial system, leads to hepatosplenomegaly, anemia and skeletal lesions in type I disease, and neurologic manifestations in types II and III disease. We report herein the identification of hydrophilic active-site-specific chaperones that are capable of increasing glucocerebrosidase activity in the cultured fibroblasts of Gaucher patients. Screening of a variety of natural and synthetic alkaloid compounds showed isofagomine, N-dodecyl deoxynojirimycin, calystegines A 3 , B 1 , B 2 and C 1 , and 1,5-dideoxy-1,5-iminoxylitol to be potent inhibitors of glucocerebrosidase. Among them, isofagomine was the most potent inhibitor of glucocerebrosidase in vitro, and the most effective active-site-specific chaperone capable of increasing residual glucocerebrosidase activity in fibroblasts established from Gaucher patients with the most prevalent Gaucher disease-causing mutation (N370S). Intracellular enzyme activity increased approximately two-fold after cells had been incubated with isofagomine, and the increase in glucocerebrosidase activity was both dose-dependent and time-dependent. Western blotting demonstrated that there was a substantial increase in glucocerebrosidase protein in cells after isofagomine treatment. Immunocytochemistry revealed an improvement in the glucocerebrosidase trafficking pattern, which overlaps that of lysosome-associated membrane protein 2 in Gaucher fibroblasts cultivated with isofagomine, suggesting that the transport of mutant glucocerebrosidase is at least partially improved in the presence of isofagomine. The hydrophilic active-site-specific chaperones are less toxic to cultured cells. These results indicate that these hydrophilic small molecules are suitable candidates for further drug development for the treatment of Gaucher disease.

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.…”

“…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

“…[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.…”

Identification of Potent and Selective Glucosylceramide Synthase Inhibitors from a Library of N-Alkylated Iminosugars

“…A similar trend is seen in the L-ido-configured series. Again, GCS inhibitory potency increases with increasing N-alkyl substituent, and again, the N-alkyloxyalkyl derivatives (22)(23)(24)(25)(26)(27) outperform the N-alkyl species (10)(11)(12)(13)(14)(15). Head-to-head comparison of two stereoisomers from the two series reveals that in general the L-ido congener is the most potent GCS inhibitor.…”

“…[14][15][16][17][18][19] Potent and selective GCS inhibitors thus hold considerable potential therapeutic value, and the development of such compounds is pursued by an increasing number of researchers worldwide. [20][21][22][23][24][25][26][27][28][29][30] In the search for selective GCS inhibitors, we give particular attention to the two known hydrolytic activities capable of hydrolyzing glucosylceramide, next to the aforementioned GBA1 and also the nonlysosomal glucosylceramidase or GBA2. As said, partially dysfunctional GBA1 is at the basis of Gaucher DOI: 10.1021/ml100192b |ACS Med.…”