Preclinical efficacy and safety of 1-deoxygalactonojirimycin in mice for Fabry disease.

Fabry disease is an inborn error of glycosphingolipid metabolism caused by deficiency of alpha-galactosidase A (alpha-Gal A) activity. It has been shown that protein misfolding is primarily responsible for the enzyme deficiency in a large proportion of mutations identified in Fabry patients with residual enzyme activity, and 1-deoxygalactonojirimycin (DGJ) can effectively increase the residual enzyme activity in cultured patient's cells. Herein, we demonstrate the preclinical efficacy and safety of DGJ in transgenic mice that express human mutant alpha-Gal A activity. alpha-Gal A activity in heart, kidney, spleen, and liver was increased dose- and time-dependently. The mutant alpha-Gal A was increased in cardiomyocytes and distal convoluted tubules of the transgenic mice in a null background after 2 weeks of DGJ treatment. Globotriaosylceramide storage was remarkably reduced in kidney of mice after a 4-week treatment at a dosage of approximately 3 mg/kg body weight/day. The half-life of DGJ was less than 1 day in all major issues and that of the enzyme synthesized during the DGJ treatment period was approximately 4 days. No abnormality of blood chemistry and pathological tissue damage was found in mice treated with DGJ at approximately 30 mg/kg body weight/day for 9 weeks. Furthermore, no change was observed in appearance, growth, fertility, and life span in mice during a 2-year period of continuous administration of DGJ at the effective dosage. These preclinical results indicate that DGJ is effective in restoring mutant enzyme activity in tissues and reversing substrate storage in kidney and is well tolerated in mice.

Rescue of mutant alpha-galactosidase A in the endoplasmic reticulum by 1-deoxygalactonojirimycin leads to trafficking to lysosomes.

Active-site-specific chaperone therapy for Fabry disease is a genotype-specific therapy using a competitive inhibitor, 1-deoxygalactonojirimycin (DGJ). To elucidate the mechanism of enhancing alpha-galactosidase A (alpha-Gal A) activity by DGJ-treatment, we studied the degradation of a mutant protein and the effect of DGJ in the endoplasmic reticulum (ER). We first established an in vitro translation and translocation system using rabbit reticulocyte lysates and canine pancreas microsomal vesicles for a study on the stability of mutant alpha-Gal A with an amino acid substitution (R301Q) in the ER. R301Q was rapidly degraded, but no degradation of wild-type alpha-Gal A was observed when microsomal vesicles containing wild-type or R301Q alpha-Gal A were isolated and incubated. A pulse-chase experiment on R301Q-expressing TgM/KO mouse fibroblasts showed rapid degradation of R301Q, and its degradation was blocked by the addition of lactacystin, indicating that R301Q was degraded by ER-associated degradation (ERAD). Rapid degradation of R301Q was also observed in TgM/KO mouse fibroblasts treated with brefeldin A, and the amount of R301Q enzyme markedly increased by pretreatment with DGJ starting 12 h prior to addition of brefeldin A. The enhancement of alpha-Gal A activity and its protein level by DGJ-treatment was selectively observed in brefeldin A-treated COS-7 cells expressing R301Q but not in cells expressing the wild-type alpha-Gal A. Observation by immunoelectron microscopy showed that the localization of R301Q in COS-7 cells was in the lysosomes, not the ER. These data suggest that the rescue of R301Q from ERAD is a key step for normalization of intracellular trafficking of R301Q.

A counterintuitive approach to treat enzyme deficiencies: use of enzyme inhibitors for restoring mutant enzyme activity.

Pharmacological chaperone therapy is an emerging counterintuitive approach to treat protein deficiencies resulting from mutations causing misfolded protein conformations. Active-site-specific chaperones (ASSCs) are enzyme active-site directed small molecule pharmacological chaperones that act as a folding template to assist protein folding of mutant proteins in the endoplasmic reticulum (ER). As a result, excessive degradation of mutant proteins in the ER-associated degradation (ERAD) machinery can be prevented, thus restoring enzyme activity. Lysosomal storage disorders (LSDs) are suitable candidates for ASSC treatment, as the levels of enzyme activity needed to prevent substrate storage are relatively low. In addition, ASSCs are orally active small molecules and have potential to gain access to most cell types to treat neuronopathic LSDs. Competitive enzyme inhibitors are effective ASSCs when they are used at sub-inhibitory concentrations. This whole new paradigm provides excellent opportunity for identifying specific drugs to treat a broad range of inherited disorders. This review describes protein misfolding as a pathophysiological cause in LSDs and provides an overview of recent advances in the development of pharmacological chaperone therapy for the diseases. In addition, a generalized guidance for the design and screening of ASSCs is also presented.

Active-site-specific chaperone therapy for Fabry disease. Yin and Yang of enzyme inhibitors.

Protein misfolding is recognized as an important pathophysiological cause of protein deficiency in many genetic disorders. Inherited mutations can disrupt native protein folding, thereby producing proteins with misfolded conformations. These misfolded proteins are consequently retained and degraded by endoplasmic reticulum-associated degradation, although they would otherwise be catalytically fully or partially active. Active-site directed competitive inhibitors are often effective active-site-specific chaperones when they are used at subinhibitory concentrations. Active-site-specific chaperones act as a folding template in the endoplasmic reticulum to facilitate folding of mutant proteins, thereby accelerating their smooth escape from the endoplasmic reticulum-associated degradation to maintain a higher level of residual enzyme activity. In Fabry disease, degradation of mutant lysosomal alpha-galactosidase A caused by a large set of missense mutations was demonstrated to occur within the endoplasmic reticulum-associated degradation as a result of the misfolding of mutant proteins. 1-Deoxygalactonojirimycin is one of the most potent inhibitors of alpha-galactosidase A. It has also been shown to be the most effective active-site-specific chaperone at increasing residual enzyme activity in cultured fibroblasts and lymphoblasts established from Fabry patients with a variety of missense mutations. Oral administration of 1-deoxygalactonojirimycin to transgenic mice expressing human R301Q alpha-galactosidase A yielded higher alpha-galactosidase A activity in major tissues. These results indicate that 1-deoxygalactonojirimycin could be of therapeutic benefit to Fabry patients with a variety of missense mutations, and that the active-site-specific chaperone approach using functional small molecules may be broadly applicable to other lysosomal storage disorders and other protein deficiencies.

Mutant alpha-galactosidase A enzymes identified in Fabry disease patients with residual enzyme activity: biochemical characterization and restoration of normal intracellular processing by 1-deoxygalactonojirimycin.

Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.

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

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 A3, B1, B2 and C1, 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.

Transgenic mouse expressing human mutant alpha-galactosidase A in an endogenous enzyme deficient background: a biochemical animal model for studying active-site specific chaperone therapy for Fabry disease.

Fabry disease is an inborn error of glycosphingolipid metabolism caused by the deficiency of lysosomal alpha-galactosidase A (alpha-Gal A). We have established transgenic mice that exclusively express human mutant alpha-Gal A (R301Q) in an alpha-Gal A knock-out background (TgM/KO mice). This serves as a biochemical model to study and evaluate active-site specific chaperone (ASSC) therapy for Fabry disease, which is specific for those missense mutations that cause misfolding of alpha-Gal A. The alpha-Gal A activities in the heart, kidney, spleen, and liver of homozygous TgM/KO mice were 52.6, 9.9, 29.6 and 44.4 unit/mg protein, respectively, corresponding to 16.4-, 0.8-, 0.6- and 1.4-fold of the endogenous enzyme activities in the same tissues of non-transgenic mice with a similar genetic background. Oral administration of 1-deoxygalactonojirimycin (DGJ), a competitive inhibitor of alpha-Gal A and an effective ASSC for Fabry disease, at 0.05 mM in the drinking water of the mice for 2 weeks resulted in 13.8-, 3.3-, 3.9-, and 2.6-fold increases in enzyme activities in the heart, kidney, spleen and liver, respectively. No accumulation of globotriaosylceramide, a natural substrate of alpha-Gal A, could be detected in the heart of TgM/KO mice after DGJ treatment, indicating that degradation of the glycolipid in the heart was not inhibited by DGJ at that dosage. The alpha-Gal A activity in homozygous or heterozygous fibroblasts established from TgM/KO mice (TMK cells) was approximately 39 and 20 unit/mg protein, respectively. These TgM/KO mice and TMK cells are useful tools for studying the mechanism of ASSC therapy, and for screening ASSCs for Fabry disease.

Efficient and rapid purification of recombinant human alpha-galactosidase A by affinity column chromatography.

The lysosomal enzyme alpha-galactosidase A (alpha-Gal A) metabolizes neutral glycosphingolipids that possess alpha-galactoside residues at the non-reducing terminus, and inherited defects in the activity of alpha-Gal A lead to Fabry disease. We describe here an efficient and rapid purification procedure for recombinant alpha-Gal A by sequential Concanavalin A (Con A)-Sepharose and immobilized thio-alpha-galactoside (thio-Gal) agarose column chromatography. Optimal elution conditions for both columns were obtained using overexpressed human alpha-Gal A. We recommend the use of a mixture of 0.9 M methyl alpha-mannoside and 0.9 M methyl alpha-glucoside in 0.1 M acetate buffer (pH 6.0) with 0.1 M NaCl for the maximum recovery of glycoproteins with multiple high-mannose type sugar chains from Con A column chromatography, and that the Con A column should not be reused for the purification of glycoproteins that are used for structural studies. Binding of the enzyme to the thio-Gal column requires acidic condition at pH 4.8. A galactose-containing buffer (25 mM citrate-phosphate buffer, pH 5.5, with 0.1 M galactose, and 0.1 M NaCl) was used to elute alpha-Gal A. This procedure is especially useful for the purification of mutant forms of alpha-Gal A, which are not stable under conventional purification techniques. A protocol that purifies an intracellular mutant alpha-Gal A (M279I) expressed in COS-7 cells within 6h at 62% overall yield is presented.

Alternative splicing in the alpha-galactosidase A gene: increased exon inclusion results in the Fabry cardiac phenotype.

Fabry disease is an inborn error of glycosphingolipid catabolism, resulting from deficient activity of lysosomal alpha-galactosidase A (alpha-Gal A). A rare alternative splicing that introduces a 57-nucleotide (nt) intronic sequence to the alpha-Gal A transcript from intron 4 of the gene has been identified. In addition, a novel midintronic base substitution that results in substantially increased alternative splicing has been identified in a patient with Fabry disease who has the cardiac variant phenotype. The sequence of the patient's intron 4 contains a single G-->A transversion at genomic nt 9331 (IVS4+919 G-->A ), located at the minus sign4 position of the 3' end of the intronic insertion (nts 9278--9334 in the genomic sequence). Minigene constructs containing the entire intron 4 sequence with G, A, C, or T at nt 9331 within an alpha-Gal A complementary DNA expression vector were prepared and expressed in COS-1 cells. Whereas transfection of the G or T minigenes transcribed predominantly normal-sized transcripts, the transfection of the A or C minigenes produced a large amount of the alternatively spliced transcript. These results suggest that the G-->A mutation, within an A/C-rich domain, results in increased recognition of the alternative splicing by an A/C-rich enhancer-type exonic splicing enhancer. The intronic mutation was not observed in 100 unrelated unaffected men but was present in 6 unrelated patients with cardiac Fabry disease. Reverse-transcriptase polymerase chain reaction of total RNA of various normal human tissues revealed that the alternatively spliced transcript was present in all of the samples, and especially at a higher ratio in the lung and muscle. The normal transcript was present in the patients' lymphoblasts and resulted in approximately 10% residual enzyme activity, leading to a cardiac phenotype of Fabry disease.