In vitro inhibition and intracellular enhancement of lysosomal α‐galactosidase A activity in Fabry lymphoblasts by 1‐deoxygalactonojirimycin and its derivatives

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Abstract
Fabry disease is a lysosomal storage disorder caused by deficient lysosomal α‐galactosidase A (α‐Gal A) activity. Deficiency of the enzyme activity results in progressive deposition of neutral glycosphingolipids with terminal α‐galactosyl residue in vascular endothelial cells. We recently proposed a chemical chaperone therapy for this disease by administration of 1‐deoxygalactonojirimycin, a potent inhibitor of the enzyme, at subinhibitory intracellular concentrations [Fan, J.‐Q., Ishii, S., Asano, N. and Suzuki, Y. (1999) Nat. Med.5, 112–115]. 1‐Deoxygalactonojirimycin served as a specific chaperone for those mutant enzymes that failed to maintain their proper conformation to avoid excessive degradation. In order to establish a correlation between in vitro inhibitory activity and intracellular enhancement activity of the specific chemical chaperone, a series of 1‐deoxygalactonojirimycin derivatives were tested for activity with both α‐Gal A and Fabry lymphoblasts. 1‐Deoxygalactonojirimycin was the most potent inhibitor of α‐Gal A with an IC50 value of 0.04 µm. α‐Galacto‐homonojirimycin, α‐allo‐homonojirimycin and β‐1‐C‐butyl‐deoxygalactonojirimycin were effective inhibitors with IC50 values of 0.21, 4.3 and 16 µm, respectively. N‐Alkylation, deoxygenation at C‐2 and epimerization at C‐3 of 1‐deoxygalactonojirimycin markedly lowered or abolished its inhibition toward α‐Gal A. Inclusion of 1‐deoxygalactonojirimycin, α‐galacto‐homonojirimycin, α‐allo‐homonojirimycin and β‐1‐C‐butyl‐deoxygalactonojirimycin at 100 µm in culture medium of Fabry lymphoblasts increased the intracellular α‐Gal A activity by 14‐fold, 5.2‐fold, 2.4‐fold and 2.3‐fold, respectively. Weaker inhibitors showed only a minimum enhancement effect. These results suggest that more potent inhibitors act as more effective specific chemical chaperones for the mutant enzyme, and the potent competitive inhibitors of α‐Gal A are effective specific chemical chaperones for Fabry disease.