piperidines and Fabry-Disease

Metabolic storage disorders are caused by mutations in genes that result in insufficient activity of enzymes required for the catabolism of substances that arise from the turnover of senescent cells in the body. Among the most prevalent of these conditions are Gaucher disease and Fabry disease, which are caused by reduced activity of the housekeeping enzymes glucocerebrosidase and alpha-galactosidase A, respectively. Enzyme replacement therapy is extraordinarily effective for patients with Gaucher disease. It is under examination in patients with Fabry disease, and improvement of various clinical aspects in these patients has been documented. The blood-brain barrier prevents systemically administered enzymes from reaching the central nervous system. This limitation is a major impediment for the treatment of patients with enzyme deficiency disorders in whom the brain is involved. Alternatives to enzyme replacement therapy that have been initiated to treat systemic manifestations and brain involvement in patients with metabolic disorders include substrate reduction therapy, active site-specific chaperone therapy, and gene therapy. The present status and anticipated advances in the application of these therapeutic approaches are examined here.

Topics: 1-Deoxynojirimycin; Bone Marrow Transplantation; Enzyme Inhibitors; Fabry Disease; Gangliosidosis, GM1; Gaucher Disease; Genetic Therapy; Humans; Imino Sugars; Piperidines

Topics: 1-Deoxynojirimycin; alpha-Galactosidase; Animals; Enzyme Stability; Fabry Disease; Galactose; Gaucher Disease; Glycoside Hydrolases; Humans; Imino Sugars; Molecular Weight; Piperidines; Protein Conformation; Protein Folding

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.

Topics: alpha-Galactosidase; Cell Line; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme Stability; Fabry Disease; Humans; Molecular Structure; Piperidines; Recombinant Proteins; Structure-Activity Relationship

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.

Topics: alpha-Galactosidase; Campanulaceae; Fabry Disease; Humans; Imino Sugars; Molecular Chaperones; Mutant Proteins; Phytotherapy; Piperidines; Plant Extracts; Protein Transport; Pyrrolidines; Sugar Alcohols

Fabry disease is a lysosomal storage disorder caused by deficiency of alpha-galactosidase A (alpha-Gal A) resulting in lysosomal accumulation of glycosphingolipid globotriosylceramide Gb3. Misfolded alpha-Gal A variants can have residual enzyme activity but are unstable. Their lysosomal trafficking is impaired because they are retained in the endoplasmic reticulum (ER) by quality control. Subinhibitory doses of the competitive inhibitor of alpha-Gal A, 1-deoxygalactonojirimycin (DGJ), stabilize mutant alpha-Gal A in vitro and correct the trafficking defect. We showed by immunolabeling that the chaperone-like action of DGJ significantly reduces the lysosomal Gb3 storage in human Fabry fibroblasts harboring the novel mutations T194I and V390fsX8. The specificity of the DGJ effect was proven by RNA interference. Electron microscopic morphometry demonstrated a reduction of large-size, disease-associated lysosomes and loss of characteristic multilamellar lysosomal inclusions on DGJ treatment. In addition, the pre-Golgi intermediates were decreased. However, the rough ER was not different between DGJ-treated and untreated cells. Pulse-chase experiments revealed that DGJ treatment resulted in maturation and stabilization of mutant alpha-Gal A. Genes involved in cell stress signaling, heat shock response, unfolded protein response, and ER-associated degradation show no apparent difference in expression between untreated and DGJ-treated fibroblasts. The DGJ treatment has no apparent cytotoxic effects. Thus our data show the usefulness of a pharmacological chaperone for correction of the lysosomal storage in Fabry fibroblasts harboring different mutations with residual enzyme activity. Pharmacological chaperones acting on misfolded, unstable mutant proteins that exhibit residual biological activity offer a convenient and cost-efficient therapeutic strategy.

Topics: 1-Deoxynojirimycin; alpha-Galactosidase; Cells, Cultured; DNA Mutational Analysis; Enzyme Stability; Fabry Disease; Fibroblasts; Gene Expression; Humans; Imino Sugars; Lysosomes; Male; Molecular Chaperones; Piperidines; Protein Transport; RNA Interference