Inhibition of melanoma tumor growth by a novel inhibitor of glucosylceramide synthase. (1/21)

Tumor ganglioside metabolism has been implicated in modulating tumor formation and progression. We found previously that transient ganglioside depletion by inhibition of glucosylceramide synthesis of MEB4 melanoma cells in vitro reduced their tumorigenic capability. Here, we have established that treatment of the host with a novel p.o. inhibitor of glucosylceramide synthesis, the imino sugar OGT2378, inhibits MEB4 melanoma tumor growth in a syngeneic, orthotopic murine model. The glucosylceramide and ganglioside content of MEB4 cells exposed to 20 micro M OGT2378 in culture were reduced by 93 and >95%, respectively, without either cytotoxic or antiproliferative effects. Administered in the diet to C57BL/6 mice, 2500 mg/kg/day OGT2378 was well tolerated in vivo and biologically active, depleting host tissue (hepatic) gangliosides by 82% and tumor gangliosides by >98%. p.o. treatment with OGT2378 starting 3 days before intradermal tumor inoculation of 4 x 10(4) MEB4 cells, and continuing for 4 weeks, resulted in a 10-fold lower mean tumor volume at the end of treatment (60 versus 538 mm(3), P < 0.0001). Even when OGT2378 treatment was initiated 7 days after tumor inoculation, tumor growth was similarly impeded (61 versus 620 mm(3), P < 0.0001), demonstrating an effect on an established tumor. The effectiveness of p.o. OGT2378 in this murine model suggests that inhibition of glycosphingolipid synthesis is a promising and now feasible novel therapeutic approach to inhibit tumor progression.  (+info)

The alkylated imino sugar, n-(n-Nonyl)-deoxygalactonojirimycin, reduces the amount of hepatitis B virus nucleocapsid in tissue culture. (2/21)

n-(n-Nonyl)-deoxygalactonojirimycin (n,n-DGJ), an alkylated imino sugar, reduces the amount of HBV DNA produced within the stably transfected HBV-producing HepG2.2.15 line in culture and is under consideration for development as a human therapeutic. n,n-DGJ does not appear to inhibit HBV DNA polymerase activity or envelop antigen production (A. Mehta, S. Carrouee, B. Conyers, R. Jordan, T. Butters, R. A. Dwek, and T. M. Block, Hepatology 33:1488-1495, 2001), and the mechanism of antiviral action is unknown. In this study, the step in the virus life cycle affected by n,n-DGJ was explored. Using Northern analysis and immunoprecipitation with anti-HBc antibody, we found that, under conditions in which cell viability was not affected but viral DNA production was substantially reduced, neither the amount of HBV transcription products nor the core polypeptide was detectably reduced. However, the pregenomic RNA, endogenous polymerase activity, and core polypeptide sedimenting in sucrose gradients with a density consistent with that of assembled nucleocapsids were significantly less in the HepG2.2.15 cells incubated with n,n-DGJ. These data suggest that n,n-DGJ either prevents the maturation of HBV nucleocapsids or destabilizes the formed nucleocapsids. Although the cellular and viral mediators of this inhibition are not known, depletion of nucleocapsid has been attributed to some other compounds as well as interferon's mechanism of anti-HBV action. The similarities and differences between this alkylated imino sugar and these other mediators are discussed.  (+info)

Inhibition of glucosylceramide synthase does not reverse drug resistance in cancer cells. (3/21)

The multidrug-resistant cancer cell lines NCI/AdR(RES) and MES-SA/DX-5 have higher glycolipid levels and higher P-glycoprotein expression than the chemosensitive cell lines MCF7-wt and MES-SA. Inhibiting glycolipid biosynthesis by blocking glucosylceramide synthase has been proposed to reverse drug resistance in MDR cells by causing an increased accumulation of proapoptotic ceramide during treatment of cells with cytotoxic drugs. We treated both multidrug-resistant cell lines with the glucosylceramide synthase inhibitors PDMP (d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol), C9DGJ (N-nonyl-deoxygalactonojirimycin) or C4DGJ (N-butyl-deoxygalactonojirimycin). PDMP achieved a significant reversal of drug resistance in agreement with previous reports. However, the N-alkylated iminosugars C9DGJ and C4DGJ, which are more selective glucosylceramide synthase inhibitors than PDMP, failed to cause any reversal of drug resistance despite depleting glycolipids to the same extent as PDMP. Our results suggest that (a) inhibition of glucosylceramide synthase does not reverse multidrug resistance and (b) the chemosensitization achieved by PDMP cannot be caused by inhibition of glucosylceramide synthase alone.  (+info)

Imino sugar inhibitors for treating the lysosomal glycosphingolipidoses. (4/21)

The inherited metabolic disorders of glycosphingolipid (GSL) metabolism are a relatively rare group of diseases that have diverse and often neurodegenerative phenotypes. Typically, a deficiency in catabolic enzyme activity leads to lysosomal storage of GSL substrates and in many diseases, several other glycoconjugates. A novel generic approach to treating these diseases has been termed substrate reduction therapy (SRT), and the discovery and development of N-alkylated imino sugars as effective and approved drugs is discussed. An understanding of the molecular mechanism for the inhibition of the key enzyme in GSL biosynthesis, ceramide glucosyltransferase (CGT) by N-alkylated imino sugars, has also lead to compound design for improvements to inhibitory potency, bioavailability, enzyme selectivity, and biological safety. Following a successful clinical evaluation of one compound, N-butyl-deoxynojirimycin [(NB-DNJ), miglustat, Zavesca], for treating type I Gaucher disease, issues regarding the significance of side effects and CNS access have been addressed as exposure of drug to patients has increased. An alternative experimental approach to treat specific glycosphingolipid (GSL) lysosomal storage diseases is to use imino sugars as molecular chaperons that assist protein folding and stability of mutant enzymes. The principles of chaperon-mediated therapy (CMT) are described, and the potential efficacy and preclinical status of imino sugars is compared with substrate reduction therapy (SRT). The increasing use of imino sugars for clinical evaluation of a group of storage diseases that are complex and often intractable disorders to treat has considerable benefit. This is particularly so given the ability of small molecules to be orally available, penetrate the central nervous system (CNS), and have well-characterized biological and pharmacological properties.  (+info)

Long-term non-hormonal male contraception in mice using N-butyldeoxynojirimycin. (5/21)

BACKGROUND: The imino sugar N-butyldeoxynojirimycin (NB-DNJ) causes reversible infertility in male mice. This compound may have promise as a male contraceptive, because it is already in clinical use, for a non-reproductive condition. As contraceptives need to be taken for extended periods of time, it was essential to evaluate NB-DNJ for its reproductive effects over a long period of administration. METHODS: We have assessed the imino sugar for its long-term effects on the fertility of male C57BL/6 mice, reversibility and potential cumulative toxicity by monitoring various reproductive and systemic parameters over 12 months. RESULTS: Long-term low-dose (15 mg/kg/day) administration of NB-DNJ was sufficient to maintain infertility in male mice. In contrast to short-term drug treatment, imino sugar exposure for more than 3 months resulted in reduced sperm counts. Male mice that had been administered imino sugar for 6, 10 or 12 months and were then maintained without drug administration regained their fertility within 9 weeks after withdrawal of the drug. Prolonged NB-DNJ intake did not affect reproductive hormone levels, serum biochemistry or animal behaviour. CONCLUSION: Low-dose treatment with NB-DNJ over a long period is an effective approach for the regulation of fertility in a male mammal by non-hormonal means, without causing overt adverse effects.  (+info)

Pharmacological chaperone corrects lysosomal storage in Fabry disease caused by trafficking-incompetent variants. (6/21)

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.  (+info)

Imino sugars are potent agonists of the human glucose sensor SGLT3. (7/21)

Imino sugars are used to treat type 2 diabetes mellitus [miglitol (Glyset)] and lysosomal storage disorders [miglustat (Zavesca)] based on the inhibition of alpha-glucosidases and glucosyltransferases. In this substrate specificity study, we examined the interactions of imino sugars with a novel human glucose sensor, sodium/glucose cotransporter type 3 (hSGLT3), using expression in Xenopus laevis oocytes and electrophysiology. The results for hSGLT3 are compared with those for alpha-glucosidases and human SGLT type 1 (hSGLT1), a well characterized sodium/glucose cotransporter of the SGLT family. In general, substrates have lower apparent affinities (K0.5) for hSGLT3 than hSGLT1 (D-glucose, alpha-methyl-D-glucose, 1-deoxy-D-glucose, and 4-deoxy-4-fluoro-D-glucose exhibit K0.5 values of 19, 21, 43, and 17 mM, respectively, for hSGLT3, and 0.5, 0.7, 10, and 0.07 mM, respectively, for hSGLT1). However, specificity of hSGLT3 binding is greater (D-galactose and 4-deoxy-4-fluoro-D-galactose are not hSGLT3 substrates, but have hSGLT1 K0.5 values of 0.6 and 1.3 mM). An important deviation from this trend is potent hSGLT3 activation by the imino sugars 1-deoxynojirimycin (DNJ), N-hydroxylethyl-1-deoxynojirimycin (miglitol), N-butyl-1-deoxynojirimycin (miglustat), N-ethyl-1-deoxynojirimycin, and 1-deoxynojirimycin-1-sulfonic acid, with K0.5 values of 0.5 to 9 microM. The diastereomer 1-deoxygalactonojirimycin activates hSGT3 with a K0.5 value of 11 mM, a 3000-fold less potent interaction than is observed for DNJ (4 microM). These imino sugar binding characteristics are similar to those for alpha-glucosidases, but there are no interactions with hSGLT1. This work provides insights into hSGLT3 and -1 substrate binding interactions, establishes a pharmacological profile to study endogenous hSGLT3, and may have important ramifications for the clinical application of imino sugars.  (+info)

Accumulation of glucosylceramide in murine testis, caused by inhibition of beta-glucosidase 2: implications for spermatogenesis. (8/21)

One of the hallmarks of male germ cell development is the formation of a specialized secretory organelle, the acrosome. This process can be pharmacologically disturbed in C57BL/6 mice, and thus infertility can be induced, by small molecular sugar-like compounds (alkylated imino sugars). Here the biochemical basis of this effect has been investigated. Our findings suggest that in vivo alkylated imino sugars primarily interact with the non-lysosomal glucosylceramidase. This enzyme cleaves glucosylceramide into glucose and ceramide, is sensitive to imino sugars in vitro, and has been characterized as beta-glucosidase 2 (GBA2). Imino sugars raised the level of glucosylceramide in brain, spleen, and testis, in a dose-dependent fashion. In testis, multiple species of glucosylceramide were similarly elevated, those having long acyl chains (C16-24), as well as those with very long polyunsaturated acyl chains (C28-30:5). Both of these GlcCer species were also increased in the testes from GBA2-deficient mice. When considering that the very long polyunsaturated sphingolipids are restricted to germ cells, these results indicate that in the testis GBA2 is present in both somatic and germ cells. Furthermore, in all mouse strains tested imino sugar treatment caused a rise in testicular glucosylceramide, even in a number of strains, of which the males remain fertile after drug administration. Therefore, it appears that acrosome formation can be derailed by accumulation of glucosylceramide in an extralysosomal localization, and that the sensitivity of male germ cells to glucosylceramide is genetically determined.  (+info)

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