Synthesis of a New β-Galactosidase Inhibitor Displaying Pharmacological Chaperone Properties for GM1 Gangliosidosis.
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ABSTRACT:
SUBMITTER: Clemente F
PROVIDER: S-EPMC9268699 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Lysosomal β-galactosidase (β-Gal) deficiency causes a group of disorders that include neuronopathic GM1 gangliosidosis and non-neuronopathic Morquio B disease. We have previously proposed the use of small molecule ligands of β-Gal as pharmacological chaperones (PCs) for the treatment of GM1 gangliosidosis brain pathology. Although it is still under development, PC therapy has yielded promising preclinical results in several lysosomal diseases. In this study, we evaluated the effect of bicyclic 1-deoxygalactonojirimycin (DGJ) derivative of the sp(2)-iminosugar type, namely 5N,6S-(N'-butyliminomethylidene)-6-thio-1- deoxygalactonojirimycin (6S-NBI-DGJ), as a novel PC for human mutant β-Gal. In vitro, 6S-NBI-DGJ had the ability to inhibit the activity of human β-Gal in a competitive manner and was able to protect this enzyme from heat-induced degradation. Computational analysis supported that the rigid glycone bicyclic core of 6S-NBI-DGJ binds to the active site of the enzyme, with the aglycone N'-butyl substituent, in a precise E-orientation, located at a hydrophobic region nearby. Chaperone potential profiling indicated significant increases of enzyme activity in 24 of 88 β-Gal mutants, including four common mutations. Finally, oral administration of 6S-NBI-DGJ ameliorated the brain pathology of GM1 gangliosidosis model mice. These results suggest that 6S-NBI-DGJ is a novel PC that may be effective on a broad range of β-Gal mutants.
| S-EPMC3589148 | biostudies-literature
Project description:G(M1) gangliosidosis and Morquio B are autosomal recessive lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme β-d-galactosidase (β-Gal), which lead to accumulations of the β-Gal substrates, G(M1) ganglioside, and keratan sulfate. β-Gal is an exoglycosidase that catalyzes the hydrolysis of terminal β-linked galactose residues. This study shows the crystal structures of human β-Gal in complex with its catalytic product galactose or with its inhibitor 1-deoxygalactonojirimycin. Human β-Gal is composed of a catalytic TIM barrel domain followed by β-domain 1 and β-domain 2. To gain structural insight into the molecular defects of β-Gal in the above diseases, the disease-causing mutations were mapped onto the three-dimensional structure. Finally, the possible causes of the diseases are discussed.
| S-EPMC3265862 | biostudies-literature
Project description:Autosomal recessive mutations in the galactosidase β1 (GLB1) gene cause lysosomal β-gal deficiency, resulting in accumulation of galactose-containing substrates and onset of the progressive and fatal neurodegenerative lysosomal storage disease, GM1 gangliosidosis. Here, an enzyme replacement therapy (ERT) approach in fibroblasts from GM1 gangliosidosis patients with recombinant human β-gal (rhβ-gal) produced in Chinese hamster ovary cells enabled direct and precise rhβ-gal delivery to acidified lysosomes. A single, low dose (3 nm) of rhβ-gal was sufficient for normalizing β-gal activity and mediating substrate clearance for several weeks. We found that rhβ-gal uptake by the fibroblasts is dose-dependent and saturable and can be competitively inhibited by mannose 6-phosphate, suggesting cation-independent, mannose 6-phosphate receptor-mediated endocytosis from the cell surface. A single intracerebroventricularly (ICV) administered dose of rhβ-gal (100 μg) resulted in broad bilateral biodistribution of rhβ-gal to critical regions of pathology in a mouse model of GM1 gangliosidosis. Weekly ICV dosing of rhβ-gal for 8 weeks substantially reduced brain levels of ganglioside and oligosaccharide substrates and reversed well-established secondary neuropathology. Of note, unlike with the ERT approach, chronic lentivirus-mediated GLB1 overexpression in the GM1 gangliosidosis patient fibroblasts caused accumulation of a prelysosomal pool of β-gal, resulting in activation of the unfolded protein response and endoplasmic reticulum stress. This outcome was unsurprising in light of our in vitro biophysical findings for rhβ-gal, which include pH-dependent and concentration-dependent stability and dynamic self-association. Collectively, our results highlight that ICV-ERT is an effective therapeutic intervention for managing GM1 gangliosidosis potentially more safely than with gene therapy approaches.
| S-EPMC7521651 | biostudies-literature
Project description:Lysosomal beta-galactosidase (beta-Gal) occurs either alone in monomeric and dimeric forms, or in a high-M(r) complex with at least two additional proteins. One is neuraminidase and the second is the protective protein, which has also been shown to possess carboxypeptidase activity. beta-Gal activity is deficient in GM1-gangliosidosis as a primary defect, and is secondarily affected in galactosialidosis (GS), where the primary defect is the absence of protective protein activity. Fibroblasts from three patients with GM1-gangliosidosis, type 1, showed markedly reduced amounts of beta-Gal cross-reacting material (CRM), and a fourth appeared to have normal levels. A patient with type 2 GM1-gangliosidosis was also found to be CRM-normal. These findings demonstrate that patients with GM1-gangliosidosis type 1 are heterogeneous with respect to the level of residual beta-Gal protein. Fibroblasts from four patients with GS were strongly CRM-positive with an anti-beta-Gal antibody, as was a sample of brain from one of these patients, suggesting that the loss of beta-Gal activity is linked to a subtler change in the primary structure of the enzyme than has been previously thought. While three GS cell lines displayed reduced carboxypeptidase activity (to 32-42% of the control), one cell line was completely devoid of activity, demonstrating that while carboxypeptidase activity is a property of the protective protein this action is distinct and separate from its protective role. On direct immunoprecipitation with anti-beta-Gal antibody, a portion of the total carboxypeptidase activity co-precipitated with beta-Gal from extracts of normal and GM1-gangliosidosis cells, consistent with the presence of the complex in these cells. However, no carboxypeptidase activity was precipitable with this antibody from GS fibroblasts, suggesting the absence of complex from these cells. To examine this further, the various forms of beta-Gal were resolved by h.p.l.c. molecular-sieve chromatography. Three forms of beta-Gal activity were resolved in normal cells: a complex, a dimer and a monomer. Residual beta-Gal activity of GS cells resolved into two of these forms, the complex and the monomer. In normal and GM1-gangliosidosis cells a portion of the total carboxypeptidase activity co-chromatographed with the complex while the bulk of the activity occurred in a single 36,000-M(r) peak. Only the low-M(r) carboxypeptidase activity was detected in GS cells. This confirms our results on immunoprecipitation indicating that portions of the beta-Gal and the carboxypeptidase activities exist outside the complex in normal, GM1-gangliosidosis and GS cells. In summary, the loss of protective protein function from GS cells results in disproportionate loss of the dimeric and monomeric forms of beta-Gal activity, but does not result in the complete degradation of the protein.
| S-EPMC1132871 | biostudies-other
Project description:BACKGROUND: GM1 gangliosidosis (GM1) is an autosomal recessive lysosomal storage disease caused by deficiency of acid beta-galactosidase (GLB1; EC3.2.1.23). Here, we identify three novel mutations in the GLB1 gene from two Han Chinese patients with GM1 that appear correlated with clinical phenotype. METHODS: One of the two Han Chinese patients with GM1 presented with the juvenile form, and the other with the infantile form with cardiac involvement. Sequencing of the entire GLB1 gene revealed three novel mutations (p.H102 D, p.G494V, c.495_497delTCT), which were absent in 94 normal controls. Transient expression of cDNA encoding these variants was performed in COS-1 cells to evaluate ?-galactosidase activities. RESULTS: The first case (patient 1) with the juvenile form contained two missense mutations, p.H102 D and p.A301V. Patient 2 diagnosed with the infantile form of the disease with cardiac involvement was compound heterozygous for p.G494V and c.495_497delTCT mutations. All mutant beta-galactosidases exhibited significantly reduced activity (12%, 0%, 0%, and 0% for p.H102 D, p.A301V, p.G494V, and c.495_497delTCT), compared with the wild-type beta-galactosidase cDNA clone. The mutations identified in patient 2 with cardiomyopathy were localized in the GLB1 gene region common to both lysosomal beta-galactosidase and elastin binding protein (EBP), and caused a deletion in the elastin-binding domain of EBP. CONCLUSIONS: All four mutations identified in Han Chinese patients induce significant suppression of ?-galactosidase activity, correlating with severity of disease and presence of cardiomyopathy.
| S-EPMC2959015 | biostudies-literature
Project description:GM1 gangliosidosis is a neurodegenerative disorder caused by mutations in theGLB1gene, which encodes lysosomalb-galactosidase. The enzyme deficiency blocks GM1 ganglioside catabolism, leading to accumulation of GM1 ganglioside and asialo-GM1 ganglioside (GA1 glycolipid) in brain. This disease can present in varying degrees of severity, with the level of residualb-galactosidase activity primarily determining the clinical course.Glb1null mouse models, which completely lackb-galactosidase expression, exhibit a less severe form of the disease than expected from the comparable deficiency in humans, suggesting a potential species difference in the GM1 ganglioside degradation pathway. We hypothesized this difference may involve the sialidase NEU3, which acts on GM1 ganglioside to produce GA1 glycolipid. To test this hypothesis, we generatedGlb1/Neu3double knockout (DKO) mice. These mice had a significantly shorter lifespan, increased neurodegeneration, and more severe ataxia thanGlb1KO mice.Glb1/Neu3DKO mouse brains exhibited an increased GM1 ganglioside to GA1 glycolipid ratio compared withGlb1KO mice, indicating that Neu3 mediated GM1 ganglioside to GA1 glycolipid conversion inGlb1KO mice. The expression of genes associated with neuroinflammation and glial responses were enhanced inGlb1/Neu3DKO mice compared withGlb1KO mice. Mouse Neu3 more efficiently converted GM1 ganglioside to GA1 glycolipid than human NEU3 did. Our findings highlight Neu3’s role in ameliorating the consequences ofGlb1deletion in mice, provide insights into NEU3’s differential effects between mice and humans in GM1 gangliosidosis, and offer a potential therapeutic approach for reducing toxic GM1 ganglioside accumulation in GM1 gangliosidosis patients.
2023-12-14 | GSE241844 | GEO
Project description:GM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal β-galactosidase (β-Gal). The primary substrate of the enzyme is GM1-ganglioside (GM1), a sialylated glycosphingolipid abundant in nervous tissue. Patients with GM1-gangliosidosis present with massive and progressive accumulation of GM1 in the central nervous system (CNS), which leads to mental and motor decline, progressive neurodegeneration, and early death. No therapy is currently available for this lysosomal storage disease. Here, we describe a proof-of-concept preclinical study toward the development of enzyme replacement therapy (ERT) for GM1-gangliosidosis using a recombinant murine β-Gal fused to the plant lectin subunit B of ricin (mβ-Gal:RTB). We show that long-term, bi-weekly systemic injection of mβ-Gal:RTB in the β-Gal-/- mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, β-Gal activity was detected in several brain regions. This was accompanied by a reduction of accumulated GM1, reversal of neuroinflammation, and decrease in the apoptotic marker caspase 3. These results indicate that the RTB lectin delivery module enhances both the CNS-biodistribution pattern and the therapeutic efficacy of the β-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.
| S-EPMC9406850 | biostudies-literature
Project description:GM1-gangliosidosis is a lysosomal disease resulting from a deficiency in the hydrolase β-galactosidase (β-gal) and subsequent accumulation of gangliosides, primarily in neuronal tissue, leading to progressive neurological deterioration and eventually early death. Lysosomal diseases with neurological involvement have limited non-invasive therapies due to the inability of lysosomal enzymes to cross the blood-brain barrier (BBB). A novel fusion enzyme, labeled mTfR-GLB1, was designed to act as a ferry across the BBB by fusing β-gal to the mouse monoclonal antibody against the mouse transferrin receptor and tested in a murine model of GM1-gangliosidosis (β-gal-/-). Twelve hours following a single intravenous dose of mTfR-GLB1 (5.0 mg/kg) into adult β-gal-/- mice showed clearance of enzyme activity in the plasma and an increase in β-gal enzyme activity in the liver and spleen. Long-term efficacy of mTfR-GLB1 was assessed by treating β-gal-/- mice intravenously twice a week with a low (2.5 mg/kg) or high (5.0 mg/kg) dose of mTfR-GLB1 for 17 weeks. Long-term studies showed high dose mice gained weight normally compared to vehicle-treated β-gal-/- mice, which are significantly heavier than heterozygous controls. Behavioral assessment at six months of age using the pole test showed β-gal-/- mice treated with mTfR-GLB1 had improved motor function. Biochemical analysis showed an increase in β-gal enzyme activity in the high dose group from negligible levels to 20% and 11% of heterozygous levels in the liver and spleen, respectively. Together, these data show that mTfR-GLB1 is a catalytically active β-gal fusion enzyme in vivo that is readily taken up into tissues. Despite these indications of bioactivity, behavior tests other than the pole test, including the Barnes maze, inverted screen, and accelerating rotarod, showed limited or no improvement of treated mice compared to β-gal-/- mice receiving vehicle only. Further, administration of mTfR-GLB1 was insufficient to create measurable increases in β-gal enzyme activity in the brain or reduce ganglioside content (biochemically and morphologically).
| S-EPMC8025141 | biostudies-literature
Project description:GM1 gangliosidosis is a progressive, neurosomatic, lysosomal storage disorder caused by mutations in the GLB1 gene encoding the enzyme β-galactosidase. Absent or reduced β-galactosidase activity leads to the accumulation of β-linked galactose-containing glycoconjugates including the glycosphingolipid (GSL) GM1-ganglioside in neuronal tissue. GM1-gangliosidosis is classified into three forms [Type I (infantile), Type II (late-infantile and juvenile), and Type III (adult)], based on the age of onset of clinical symptoms, although the disorder is really a continuum that correlates only partially with the levels of residual enzyme activity. Severe neurocognitive decline is a feature of Type I and II disease and is associated with premature mortality. Most of the disease-causing β-galactosidase mutations reported in the literature are clustered in exons 2, 6, 15, and 16 of the GLB1 gene. So far 261 pathogenic variants have been described, missense/nonsense mutations being the most prevalent. There are five mouse models of GM1-gangliosidosis reported in the literature generated using different targeting strategies of the Glb1 murine locus. Individual models differ in terms of age of onset of the clinical, biochemical, and pathological signs and symptoms, and overall lifespan. However, they do share the major abnormalities and neurological symptoms that are characteristic of the most severe forms of GM1-gangliosidosis. These mouse models have been used to study pathogenic mechanisms, to identify biomarkers, and to evaluate therapeutic strategies. Three GLB1 gene therapy trials are currently recruiting Type I and Type II patients (NCT04273269, NCT03952637, and NCT04713475) and Type II and Type III patients are being recruited for a trial utilizing the glucosylceramide synthase inhibitor, venglustat (NCT04221451).
| S-EPMC8446533 | biostudies-literature
Project description:The lysosomal storage disorder, GM1 gangliosidosis (GM1), is a neurodegenerative condition resulting from deficiency of the enzyme β-galactosidase (β-gal). Mutation of the GLB1 gene, which codes for β-gal, prevents cleavage of the terminal β-1,4-linked galactose residue from GM1 ganglioside. Subsequent accumulation of GM1 ganglioside and other substrates in the lysosome impairs cell physiology and precipitates dysfunction of the nervous system. Beyond palliative and supportive care, no FDA-approved treatments exist for GM1 patients. Researchers are critically evaluating the efficacy of substrate reduction therapy, pharmacological chaperones, enzyme replacement therapy, stem cell transplantation, and gene therapy for GM1. A Phase I/II clinical trial for GM1 children is ongoing to evaluate the safety and efficacy of adeno-associated virus-mediated GLB1 delivery by intravenous injection, providing patients and families with hope for the future.
| S-EPMC8044076 | biostudies-literature