Fabry Disease: Molecular Basis, Pathophysiology, Diagnostics and Potential Therapeutic Directions.

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by the deficiency of α-galactosidase A (α-GalA) and the consequent accumulation of toxic metabolites such as globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). Early diagnosis and appropriate timely treatment of FD patients are crucial to prevent tissue damage and organ failure which no treatment can reverse. LSDs might profit from four main therapeutic strategies, but hitherto there is no cure. Among the therapeutic possibilities are intravenous administered enzyme replacement therapy (ERT), oral pharmacological chaperone therapy (PCT) or enzyme stabilizers, substrate reduction therapy (SRT) and the more recent gene/RNA therapy. Unfortunately, FD patients can only benefit from ERT and, since 2016, PCT, both always combined with supportive adjunctive and preventive therapies to clinically manage FD-related chronic renal, cardiac and neurological complications. Gene therapy for FD is currently studied and further strategies such as substrate reduction therapy (SRT) and novel PCTs are under investigation. In this review, we discuss the molecular basis of FD, the pathophysiology and diagnostic procedures, together with the current treatments and potential therapeutic avenues that FD patients could benefit from in the future.

Synthesis of multimeric pyrrolidine iminosugar inhibitors of human ß-glucocerebrosidase and α-galactosidase A: First example of a multivalent enzyme activity enhancer for Fabry disease.

The synthesis of a chemical library of multimeric pyrrolidine-based iminosugars by incorporation of three pairs of epimeric pyrrolidine-azides into different alkyne scaffolds via CuAAC is presented. The new multimers were evaluated as inhibitors of two important therapeutic enzymes, human α-galactosidase A (α-Gal A) and lysosomal ß-glucocerebrosidase (GCase). Structure-activity relationships were established focusing on the iminosugar inhitope, the valency of the dendron and the linker between the inhitope and the central scaffold. Remarkable is the result obtained in the inhibition of α-Gal A, where one of the nonavalent compounds showed potent inhibition (0.20 µM, competitive inhibition), being a 375-fold more potent inhibitor than the monovalent reference. The potential of the best α-Gal A inhibitors to act as pharmacological chaperones was analyzed by evaluating their ability to increase the activity of this enzyme in R301G fibroblasts from patients with Fabry disease, a genetic disorder related with a reduced activity of α-Gal A. The best enzyme activity enhancement was obtained for the same nonavalent compound, which increased 5.2-fold the activity of the misfolded enzyme at 2.5 µM, what constitutes the first example of a multivalent α-Gal A activity enhancer of potential interest in the treatment of Fabry disease.

Of the importance of the clinical phenotypes in the interpretation of the studies dealing with Fabry disease.

Fabry disease (OMIM #301500) is an X-linked disorder caused by alpha-galactosidase A deficiency with two major clinical phenotypes: classic and non-classic of different prognosis. From 2001, enzyme replacement therapies with agalsidase alfa and beta have been available. In this letter we underline the different clinical and technical considerations the readers have to be aware of to interpret the results of studies dealing with Fabry disease and anti-agalsidase antibodies. We reaffirm that antibodies preferentially develop in the severe classic Fabry phenotype, which can mislead into interpreting that antibodies are associated with much severe clinical events.

Computational and modeling approaches to understand the impact of the Fabry's disease causing mutation (D92Y) on the interaction with pharmacological chaperone 1-deoxygalactonojirimycin (DGJ).

Fabry's disease (FD) is the second most commonly occurring lysosomal storage disorders (LSDs). The mutations in α-galactosidase A (GLA) protein were widely found to be causative for the Fabry's disease. These mutations result in alternate splicing methods that affect the stability and function of the protein. The mutations near the active site of the protein results in protein misfolding. In this study, we have retrieved the missense mutation data from the three public databases (NCBI, UniProt, and HGMD). We used multiple in silico tools to predict the pathogenicity and stability of these mutations. Mutations in the active sites (D92Y, C142Y, D170V, and D266N) of the protein were screened for the phenotyping analysis using SNPeffect 4.0. Mutant D92Y was predicted to increase the amyloid propensity as well as severely reduce the protein stability and the remaining mutations showed no significant results by SNPeffect 4.0. Protein dynamics simulations (PDS) were performed to understand the behavior of the proteins due to the mutations. The simulation results showed that the D92Y mutant was more severe (higher deviation, loss of intramolecular hydrogen bonds, and lower compactness) than the other protein mutants (C142Y, D170V, and D266N). Further, the action of pharmacological chaperone 1-deoxygalactonojirimycin (DGJ) over the severe mutation was studied using the molecular docking analysis. Chaperone DGJ, an iminosugar plays a convincing role in repairing the misfolded protein and helps the protein to achieve its normal function. From the molecular docking analysis, we observed that both the native protein and protein with D92Y mutation followed similar interaction patterns. Further, the docked complexes (native-DGJ and mutant-DGJ) were subjected to PDS analysis. From the simulation analysis, we observed that DGJ had shown the better effect on the protein with the D92Y mutation. This elucidates that DGJ can still be used as a promising chaperone to treat the FD caused by mutations of GLA protein.

Deep characterization of the anti-drug antibodies developed in Fabry disease patients, a prospective analysis from the French multicenter cohort FFABRY.

BACKGROUND: Fabry disease (OMIM #301500) is an X-linked disorder caused by alpha-galactosidase A deficiency with two major clinical phenotypes: classic and non-classic of different prognosis. From 2001, enzyme replacement therapies (ERT) have been available. We aimed to determine the epidemiology and the functional characteristics of anti-drug antibodies. Patients from the French multicenter cohort FFABRY (n = 103 patients, 53 males) were prospectively screened for total anti-agalsidase IgG and IgG subclasses with a home-made enzyme-linked immunosorbent assay (ELISA), enzyme-inhibition assessed with neutralization assays and lysoGb3 plasma levels, and compared for clinical outcomes. RESULTS: Among the patients exposed to agalsidase, 40% of men (n = 18/45) and 8% of women (n = 2/25) had antibodies with a complete cross-reactivity towards both ERTs. Antibodies developed preferentially in men with non-missense GLA mutations (relative risk 2.88, p = 0.006) and classic phenotype (58.6% (17/29) vs 6.7% (1/16), p = 0.0005). Specific anti-agalsidase IgG1 were the most frequently observed (16/18 men), but the highest concentrations were observed for IgG4 (median 1.89 µg/ml, interquartile range (IQR) [0.41-12.24]). In the men exposed to agalsidase, inhibition was correlated with the total IgG titer (r = 0.67, p < 0.0001), especially IgG4 (r = 0.75, p = 0.0005) and IgG2 (r = 0.72, p = 0.001). Inhibition was confirmed intracellularly in Fabry patient leucocytes cultured with IgG-positive versus negative serum (median: 42.0 vs 75.6%, p = 0.04), which was correlated with IgG2 (r = 0.67, p = 0.017, n = 12) and IgG4 levels (r = 0.59, p = 0.041, n = 12). Plasma LysoGb3 levels were correlated with total IgG (r = 0.66, p = 0.001), IgG2 (r = 0.72, p = 0.004), IgG4 (r = 0.58, p = 0.03) and IgG1 (r = 0.55, p = 0.04) titers. Within the classic group, no clinical difference was observed but lysoGb3 levels were higher in antibody-positive patients (median 33.2 ng/ml [IQR 20.6-55.6] vs 12.5 [10.1-24.0], p = 0.005). CONCLUSION: Anti-agalsidase antibodies preferentially develop in the severe classic Fabry phenotype. They are frequently associated with enzyme inhibition and higher lysoGb3 levels. As such, they could be considered as a hallmark of severity associated with the classic phenotype. The distinction of the clinical phenotypes should now be mandatory in studies dealing with Fabry disease and its current and future therapies.

Synthesis of (3S,4S,5S)-trihydroxylpiperidine derivatives as enzyme stabilizers to improve therapeutic enzyme activity in Fabry patient cell lines.

A series of 3S,4S,5S-trihydroxylated piperidines bearing structural diversity at C-2 or C-6 positions has been synthesized and tested to determine their ability to stabilize the activity of recombinant human α-Galactosidase A (rh-α-Gal A). Hit molecules were identified by rapid inhibitory activity screening, and then further investigated for their ability to protect this enzyme from thermo-induced denaturation and enhance its activity in Fabry patient cell lines. Our study resulted in the identification of a new class of small molecules as enzyme stabilizers for the potential treatment of Fabry disease. Of these, stabilizer 21 was the most effective, showing a 12-fold increase in rh-α-Gal A activity in Fabry disease cell lines.

Investigating inhibitory activity of novel synthetic sericin peptide on α-D-glucosidase: kinetics and interaction mechanism study using a docking simulation.

BACKGROUND: We synthesised a novel sericin peptide (SP-GI) with α-d-glucosidase inhibitory activity, which has a sequence of SEDSSEVDIDLGN. The kinetics of its peptide-induced inhibition on α-d-glucosidase activity and its interaction mechanism merging with molecular docking were both investigated. RESULTS: SP-GI exhibited significant inhibitory activity with an IC50 of 2.9 ± 0.1 µmol L-1 and this inhibition was reversible and non-competitive with a Ki value of 1.0 ± 0.1 µmol L-1 . An interaction study with SP-GI revealed it bound to α-d-glucosidase at a single binding site, resulting in alterations in α-d-glucosidase secondary structure. This led to quenching of intrinsic α-d-glucosidase fluorescence by a static quenching mechanism. Molecular docking results showed that the SP-GI binding site on α-d-glucosidase differed from acarbose, with hydrogen bonding and van der Waals forces being the main binding drivers. CONCLUSION: These findings suggest the potential use for SP-GI or other natural sericin peptides as dietary supplements for the treatment of type 2 diabetes. © 2017 Society of Chemical Industry.

Rapid preparation of (3R,4S,5R) polyhydroxylated pyrrolidine-based libraries to discover a pharmacological chaperone for treatment of Fabry disease.

The rapid discovery of a pharmacological chaperone toward human α-Gal A for the treatment of Fabry disease is described. Two polyhydroxylated pyrrolidines with the (3R,4S,5R) configuration pattern underwent rapid substituent diversity by conjugating the primary aminomethyl moiety of each with a variety of carboxylic acids to generate two libraries (2 × 60 members). Our bioevaluation results showed one member with the (2R,3R,4S,5R) configuration pattern and bearing a 5-cyclohexylpentanoyl group as a substituent moiety possessed sufficient chaperoning capability to rescue α-Gal A activity in the lymphocyte of the N215S Fabry patient-derived cell line and other α-Gal A mutants in COS7 cells.

Treatment of Fabry's Disease with the Pharmacologic Chaperone Migalastat.

BACKGROUND: Fabry's disease, an X-linked disorder of lysosomal α-galactosidase deficiency, leads to substrate accumulation in multiple organs. Migalastat, an oral pharmacologic chaperone, stabilizes specific mutant forms of α-galactosidase, increasing enzyme trafficking to lysosomes. METHODS: The initial assay of mutant α-galactosidase forms that we used to categorize 67 patients with Fabry's disease for randomization to 6 months of double-blind migalastat or placebo (stage 1), followed by open-label migalastat from 6 to 12 months (stage 2) plus an additional year, had certain limitations. Before unblinding, a new, validated assay showed that 50 of the 67 participants had mutant α-galactosidase forms suitable for targeting by migalastat. The primary end point was the percentage of patients who had a response (≥50% reduction in the number of globotriaosylceramide inclusions per kidney interstitial capillary) at 6 months. We assessed safety along with disease substrates and renal, cardiovascular, and patient-reported outcomes. RESULTS: The primary end-point analysis, involving patients with mutant α-galactosidase forms that were suitable or not suitable for migalastat therapy, did not show a significant treatment effect: 13 of 32 patients (41%) who received migalastat and 9 of 32 patients (28%) who received placebo had a response at 6 months (P=0.30). Among patients with suitable mutant α-galactosidase who received migalastat for up to 24 months, the annualized changes from baseline in the estimated glomerular filtration rate (GFR) and measured GFR were -0.30±0.66 and -1.51±1.33 ml per minute per 1.73 m(2) of body-surface area, respectively. The left-ventricular-mass index decreased significantly from baseline (-7.7 g per square meter; 95% confidence interval [CI], -15.4 to -0.01), particularly when left ventricular hypertrophy was present (-18.6 g per square meter; 95% CI, -38.2 to 1.0). The severity of diarrhea, reflux, and indigestion decreased. CONCLUSIONS: Among all randomly assigned patients (with mutant α-galactosidase forms that were suitable or not suitable for migalastat therapy), the percentage of patients who had a response at 6 months did not differ significantly between the migalastat group and the placebo group. (Funded by Amicus Therapeutics; ClinicalTrials.gov numbers, NCT00925301 [study AT1001-011] and NCT01458119 [study AT1001-041].).

Looking for protein stabilizing drugs with thermal shift assay.

Thermal shift assay can be used for the high-throughput screening of pharmacological chaperones. These drugs are small molecules that bind a mutant protein and stabilize it. We demonstrated the robustness, reproducibility and versatility of the method using two molecules that are in clinical trial for Fabry or Pompe disease, Deoxygalactonojirimycin and N-Butyldeoxynojirimycin, and their target enzymes, lysosomal alpha-galactosidaseA and alpha-glucosidase, as test cases. We assessed the influence of solvents and of scanning rate on the measures. We showed that a value that is equivalent to the melting temperature can be obtained by the first derivatives of raw data. We discuss the advantages of the method and the precaution to be taken in running the experiments.

Establishing 3-nitrotyrosine as a biomarker for the vasculopathy of Fabry disease.

The endothelial dysfunction of Fabry disease results from α-galactosidase A deficiency leading to the accumulation of globotriaosylceramide. Vasculopathy in the α-galactosidase A null mouse is manifested as oxidant-induced thrombosis, accelerated atherogenesis, and impaired arterial reactivity. To better understand the pathogenesis of Fabry disease in humans, we generated a human cell model by using RNA interference. Hybrid endothelial cells were transiently transfected with small interfering RNA (siRNA) specifically directed against α-galactosidase A. Knockdown of α-galactosidase A was confirmed using immunoblotting and globotriaosylceramide accumulation. Endothelial nitric oxide synthase (eNOS) activity was correspondingly decreased by >60%. Levels of 3-nitrotyrosine (3NT), a specific marker for reactive nitrogen species and quantified using mass spectrometry, increased by 40- to 120-fold without corresponding changes in other oxidized amino acids, consistent with eNOS-derived reactive nitrogen species as the source of the reactive oxygen species. eNOS uncoupling was confirmed by the observed increase in free plasma and protein-bound aortic 3NT levels in the α-galactosidase A knockout mice. Finally, 3NT levels, assayed in biobanked plasma samples from patients with classical Fabry disease, were over sixfold elevated compared with age- and gender-matched controls. Thus, 3NT may serve as a biomarker for the vascular involvement in Fabry disease.

Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease.

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.

A Phase 2 study of migalastat hydrochloride in females with Fabry disease: selection of population, safety and pharmacodynamic effects.

BACKGROUND: Fabry disease (FD) is a genetic disorder resulting from deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) which leads to globotriaosylceramide (GL-3) accumulation in multiple tissues. We report on the safety and pharmacodynamics of migalastat hydrochloride, an investigational pharmacological chaperone given orally every other day (QOD) to females with FD. METHODS: This was an open-label, uncontrolled, Phase 2 study of 12 weeks with extension to 48 weeks in nine females with FD. Doses of 50mg, 150 mg and 250 mg were given QOD. At multiple time points, α-Gal A activity and GL-3 levels were quantified in blood cells, kidney and skin. GL-3 levels were also evaluated through skin and renal histology. Each individual GLA mutation was retrospectively categorized as being amenable or not to migalastat HCl based on an in vitro α-Gal A transfection assay developed in human embryonic kidney (HEK)-293 cells. RESULTS: Migalastat HCl was generally well tolerated. Patients with amenable mutations seem to demonstrate greater pharmacodynamic response to migalastat HCl compared to patients with non-amenable mutations. The greatest declines in urine GL-3 were observed in the three patients with amenable GLA mutations that were treated with 150 or 250 mg migalastat HCl QOD. Additionally, these three patients all demonstrated decreases in GL-3 inclusions in kidney peri-tubular capillaries. CONCLUSIONS: Migalastat HCl is a candidate oral pharmacological chaperone that provides a potential novel genotype-specific treatment for FD. Treatment resulted in GL-3 substrate decrease in female patients with amenable GLA mutations. Phase 3 studies are ongoing.

Tuning glycosidase inhibition through aglycone interactions: pharmacological chaperones for Fabry disease and GM1 gangliosidosis.

Competitive inhibitors of either α-galactosidase (α-Gal) or ß-galactosidase (ß-Gal) with high affinity and selectivity have been accessed by exploiting aglycone interactions with conformationally locked sp(2)-iminosugars. Selected compounds were profiled as potent pharmacological chaperones for mutant lysosomal α- and ß-Gal associated with Fabry disease and GM(1) gangliosidosis.

How well does urinary lyso-Gb3 function as a biomarker in Fabry disease?

BACKGROUND: Fabry disease is characterized by accumulation of glycosphingolipids, such as globotriaosylceramide (Gb(3)), in many tissues and body fluids. A novel plasma biomarker, globotriaosylsphingosine (lyso-Gb(3)), is increased in patients with the disease. Until now, lyso-Gb(3) was not detectable in urine, possibly because of the presence of interfering compounds. METHODS: We undertook to: 1) characterize lyso-Gb(3) in urine; 2) develop a method to quantitate urinary lyso-Gb(3) by mass spectrometry; 3) evaluate urinary lyso-Gb(3) as a potential biomarker for Fabry disease; and 4) determine whether lyso-Gb(3) is an inhibitor of α-galactosidase A activity. We analyzed urinary lyso-Gb(3) from 83 Fabry patients and 77 healthy age-matched controls. RESULTS: The intraday and interday bias and precision of the method were <15%. Increases in lyso-Gb(3)/creatinine correlated with the concentrations of Gb(3) (r(2)=0.43), type of mutations (p=0.0006), gender (p<0.0001) and enzyme replacement therapy status (p=0.0012). Urine from healthy controls contained no detectable lyso-Gb(3). Lyso-Gb(3) did not inhibit GLA activity in dried blood spots. Increased urinary excretion of lyso-Gb(3) of Fabry patients correlated well with a number of indicators of disease severity. CONCLUSION: Lyso-Gb(3) is a reliable independent biomarker for clinically important characteristics of Fabry disease.

2,5-Dideoxy-2,5-imino-d-altritol as a new class of pharmacological chaperone for Fabry disease.

Chromatographic separation of the extract from roots of Adenophora triphylla resulted in the isolation of two pyrrolidines, six piperidines, and two piperidine glycosides. The structures of new iminosugars were elucidated by spectroscopic methods as 2,5-dideoxy-2,5-imino-d-altritol (DIA) (2), beta-1-C-butenyl-1-deoxygalactonojirimycin (8), 2,3-dideoxy-beta-1-C-ethyl-1-deoxygalactonojirimycin (9), and 6-O-beta-d-glucopyranosyl-2,3-dideoxy-beta-1-C-ethyl-1-deoxygalactonojirimycin (10). beta-1-C-Butyl-1-deoxygalactonojirimycin (7) and compound 8 were found to be better inhibitors of alpha-galactosidase than N-butyl-1-deoxygalactonojirimycin. The present work elucidated that DIA was a powerful competitive inhibitor of human lysosome alpha-galactosidase A (alpha-Gal A) with a K(i) value of 0.5muM. Furthermore, DIA improved the thermostability of alpha-Gal A in vitro and increased intracellular alpha-Gal A activity by 9.6-fold in Fabry R301Q lymphoblasts after incubation for 3days. These experimental results suggested that DIA would act as a specific pharmacological chaperone to promote the smooth escape from the endoplasmic reticulum (ER) quality control system and to accelerate transport and maturation of the mutant enzyme.

Evaluation of antiglycosidase and anticholinesterase activities of Boehmeria nivea.

In this era, major community worldwide is suffering from diabetes type II, cancer and neurodegenerative disorders. To overcome these diseases, in the screening of Korean medicinal plants, we studied the whole plant of Boehmeria nivea (B. nivea). The methanolic leaf, stem and root extracts of B. nivea and their respective n-hexane, methylene chloride (CH(2)Cl(2)), ethyl acetate (EtOAc), n-butanol (BuOH) and aqueous fractions were investigated for their total phenolic content (TPC), 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme inhibition activities. Profound TPC and DPPH free radical scavenging activities were observed in the EtOAc and BuOH fractions of root, where the BuOH fraction showed high-pitched alpha-glucosidase inhibition and the EtOAc layer showed the maximum beta-glucosidase inhibition. Furthermore, the leaf extract demonstrated the highest beta-galactosidase inhibitory activity, but no alpha-galactosidase inhibition was seen in any of the plant parts. Notable BChE and moderate AChE inhibitory activity was found in whole plant. It can be suggested that whole plant of B. nivea provides a strong biochemical rationale as one of the good choices for the treatment of diabetes type II, cancer and neurodegenerative diseases (AD, etc).

The pharmacological chaperone 1-deoxygalactonojirimycin reduces tissue globotriaosylceramide levels in a mouse model of Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency in alpha-galactosidase A (alpha-Gal A) activity and subsequent accumulation of the substrate globotriaosylceramide (GL-3), which contributes to disease pathology. The pharmacological chaperone (PC) DGJ (1-deoxygalactonojirimycin) binds and stabilizes alpha-Gal A, increasing enzyme levels in cultured cells and in vivo. The ability of DGJ to reduce GL-3 in vivo was investigated using transgenic (Tg) mice that express a mutant form of human alpha-Gal A (R301Q) on a knockout background (Tg/KO), which leads to GL-3 accumulation in disease-relevant tissues. Four-week daily oral administration of DGJ to Tg/KO mice resulted in significant and dose-dependent increases in alpha-Gal A activity, with concomitant GL-3 reduction in skin, heart, kidney, brain, and plasma; 24-week administration resulted in even greater reductions. Compared to daily administration, less frequent DGJ administration, including repeated cycles of 4 days with DGJ followed by 3 days without or every other day with DGJ, resulted in even greater GL-3 reductions that were comparable to those obtained with Fabrazyme. Collectively, these data indicate that oral administration of DGJ increases mutant alpha-Gal A activity and reduces GL-3 in disease-relevant tissues in Tg/KO mice, and thus merits further evaluation as a treatment for Fabry disease.

Molecular interaction of imino sugars with human alpha-galactosidase: Insight into the mechanism of complex formation and pharmacological chaperone action in Fabry disease.

Enzyme enhancement therapy (EET) for Fabry disease involving imino sugars has been developed and attracted interest. It is thought that imino sugars act as pharmacological chaperones for wild-type and mutant alpha-galactosidases (GLAs) in cells, but the mechanisms underlying the molecular interactions between the imino sugars and the enzyme have not been clarified yet. We examined various kinds of imino sugars and found that galactostatin bisulfite (GBS) inhibited GLA in vitro and increased the enzyme activity in cultured Fabry fibroblasts as in the case of 1-deoxygalactonojirimycin (DGJ). Then, we analyzed the molecular interactions between the imino sugars and recombinant human GLA by means of isothermal titration calorimetry and surface plasmon resonance biosensor assays, and first determined the thermodynamic and binding-kinetics parameters of imino sugar and GLA complex formation. The results revealed that DGJ bound to the enzyme more strongly than GBS, the binding of DGJ to the enzyme protein being enthalpy-driven. In the case of GBS, the reaction was mainly enthalpy-driven, but there was a possibility that entropy-driven factors were involved in the binding. Structural analysis in silico revealed that both the chemicals fit into the active-site pocket and undergo hydrogen bonding with residues comprising the active-site pocket including the catalytic ones. The side chain of GBS was oriented towards the entrance of the active-site pocket, and thus it could be in contact with residues comprising the wall of the active-site pocket. Thermodynamic, kinetic and structural studies should provide us with a lot of information for improving EET for Fabry disease.

Reduced alpha-Gal A enzyme activity in Fabry fibroblast cells and Fabry mice tissues induced by serum from antibody positive patients with Fabry disease.

Fabry disease is a progressive, life-threatening lysosomal storage disorder which is characterized by deficient activity of the lysosomal enzyme alpha-galactosidase A. Studies have demonstrated that both enzyme preparations currently available for treatment of Fabry disease (i.e., agalsidase beta and agalsidase alpha) elicit immune responses in the majority of patients which negatively influences the reduction of urinary globotriaosylceramide concentration. In the current study, agalsidase beta antibodies were found to be associated with inhibition of alpha-Gal A enzyme activity in cultured Fabry fibroblast and tissues from Fabry mice. However, the negative effect of antibody formation could be overcome by increasing the dose of enzyme administered to mice. In conclusion, antibody titers and the dose of enzyme influenced alpha-Gal A enzyme activities in vivo. Further studies are required to investigate to what extend antibody formation impacts on therapeutic responses in antibody positive Fabry patients receiving enzyme replacement therapy and if negative effects can be overcome by adjusting the dose of enzyme.