Dataset Information

Synthesis of a New β-Galactosidase Inhibitor Displaying Pharmacological Chaperone Properties for GM1 Gangliosidosis.

ABSTRACT: GM1 gangliosidosis is a rare lysosomal disease caused by the deficiency of the enzyme β-galactosidase (β-Gal; GLB1; E.C. 3.2.1.23), responsible for the hydrolysis of terminal β-galactosyl residues from GM1 ganglioside, glycoproteins, and glycosaminoglycans, such as keratan-sulfate. With the aim of identifying new pharmacological chaperones for GM1 gangliosidosis, the synthesis of five new trihydroxypiperidine iminosugars is reported in this work. The target compounds feature a pentyl alkyl chain in different positions of the piperidine ring and different absolute configurations of the alkyl chain at C-2 and the hydroxy group at C-3. The organometallic addition of a Grignard reagent onto a carbohydrate-derived nitrone in the presence or absence of a suitable Lewis Acid was exploited, providing structural diversity at C-2, followed by the ring-closure reductive amination step. An oxidation-reduction process allowed access to a different configuration at C-3. The N-pentyl trihydroxypiperidine iminosugar was also synthesized for the purpose of comparison. The biological evaluation of the newly synthesized compounds was performed on leucocyte extracts from healthy donors and identified two suitable β-Gal inhibitors, namely compounds 10 and 12. Among these, compound 12 showed chaperoning properties since it enhanced β-Gal activity by 40% when tested on GM1 patients bearing the p.Ile51Asn/p.Arg201His mutations.

SUBMITTER: Clemente F

PROVIDER: S-EPMC9268699 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Synthesis of a New β-Galactosidase Inhibitor Displaying Pharmacological Chaperone Properties for GM1 Gangliosidosis.

Clemente Francesca F Martínez-Bailén Macarena M Matassini Camilla C Morrone Amelia A Falliano Silvia S Caciotti Anna A Paoli Paolo P Goti Andrea A Cardona Francesca F

Molecules (Basel, Switzerland) 20220622 13

GM1 gangliosidosis is a rare lysosomal disease caused by the deficiency of the enzyme β-galactosidase (β-Gal; <i>GLB1</i>; E.C. 3.2.1.23), responsible for the hydrolysis of terminal β-galactosyl residues from GM1 ganglioside, glycoproteins, and glycosaminoglycans, such as keratan-sulfate. With the aim of identifying new pharmacological chaperones for GM1 gangliosidosis, the synthesis of five new trihydroxypiperidine iminosugars is reported in this work. The target compounds feature a pentyl alky ...[more]

PMID: 35807262

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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.

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).

Dataset Information

Synthesis of a New β-Galactosidase Inhibitor Displaying Pharmacological Chaperone Properties for GM1 Gangliosidosis.

Publications

Synthesis of a New β-Galactosidase Inhibitor Displaying Pharmacological Chaperone Properties for GM1 Gangliosidosis.

Similar Datasets

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