Gabriele Reinkensmeier

University of Oxford, Oxford, England, United Kingdom

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Publications (11)106.41 Total impact

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    ABSTRACT: A series of neoglycoconjugates derived from deoxynojirimycin has been prepared by click connection with functionalised adamantanes. They have been assayed as glycosidase inhibitors, as inhibitors of the glycoenzymes relevant to the treatment of Gaucher disease, as well as correctors of the defective ion-transport protein involved in cystic fibrosis. We have demonstrated that it is possible to selectively either strongly inhibit ER-α-glucosidases and ceramide glucosyltransferase or restore the activity of CFTR in CF-KM4 cells by varying the length of the alkyl chain linking DNJ and adamantane.
    Organic & Biomolecular Chemistry 08/2011; 9(15):5373-5388. DOI:10.1039/c1ob05119a · 3.49 Impact Factor
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    ABSTRACT: Cellular α-glucosidases I and II are enzymes that sequentially trim the three terminal glucoses in the N-linked oligosaccharides of viral envelope glycoproteins. This process is essential for the proper folding of viral glycoproteins and subsequent assembly of many enveloped viruses, including dengue virus (DENV). Imino sugars are substrate mimics of α-glucosidases I and II. In this report, we show that two oxygenated alkyl imino sugar derivatives, CM-9-78 and CM-10-18, are potent inhibitors of both α-glucosidases I and II in vitro and in treated animals, and efficiently inhibit DENV infection of cultured human cells. Pharmacokinetic studies reveal that both compounds are well tolerated at doses up to 100mg/kg in rats and have favorable pharmacokinetic properties and bioavailability in mice. Moreover, we showed that oral administration of either CM-9-78 or CM-10-18 reduces the peak viremia of DENV in mice. Interestingly, while treatment of DENV infected mice with ribavirin alone did not reduce the viremia, combination therapy of ribavirin with sub-effective dose of CM-10-18 demonstrated a significantly enhanced antiviral activity, as indicated by a profound reduction of the viremia. Our findings thus suggest that combination therapy of two broad-spectrum antiviral agents may provide a practically useful approach for the treatment of DENV infection.
    Antiviral research 11/2010; 89(1):26-34. DOI:10.1016/j.antiviral.2010.11.002 · 3.61 Impact Factor
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    ABSTRACT: Highly potent N-substituted delta-lactams have been rationally designed and synthesized by a concise route with a one-pot tandem reaction as key step. These iminosugars show weak inhibition of wild-type beta-glucocerebrosidase but 3- to 6-fold increases in mutant enzyme activity (N370S).
    Journal of Medicinal Chemistry 05/2009; 52(10):3146-9. DOI:10.1021/jm801506m · 5.48 Impact Factor
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    ABSTRACT: We identified an autosomal recessive infantile-onset symptomatic epilepsy syndrome associated with developmental stagnation and blindness. Assuming a founder effect in a large Old Order Amish pedigree, we carried out a genome-wide screen for linkage and identified a single region of homozygosity on chromosome 2p12-p11.2 spanning 5.1 cM (maximum lod score of 6.84). We sequenced genes in the region and identified a nonsense mutation in SIAT9, which is predicted to result in the premature termination of the GM3 synthase enzyme (also called lactosylceramide alpha-2,3 sialyltransferase). GM3 synthase is a member of the sialyltransferase family and catalyzes the initial step in the biosynthesis of most complex gangliosides from lactosylceramide. Biochemical analysis of plasma glycosphingolipids confirmed that affected individuals lack GM3 synthase activity, as marked by a complete lack of GM3 ganglioside and its biosynthetic derivatives and an increase in lactosylceramide and its alternative derivatives. Although the relationship between defects in ganglioside catabolism and a range of lysosomal storage diseases is well documented, this is the first report, to our knowledge, of a disruption of ganglioside biosynthesis associated with human disease.
    Nature Genetics 12/2004; 36(11):1225-9. DOI:10.1038/ng1460 · 29.65 Impact Factor
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    ABSTRACT: Niemann-Pick disease type C (NP-C) is a hereditary neurovisceral lipid storage disorder. Although traditionally considered a primary cholesterol storage disorder, a variety of glycolipids accumulate in NP-C cells, which resemble those from glycosphingolipidosis patients. Substrate reduction therapy (SRT) with miglustat, an inhibitor of glycosphingolipid biosynthesis, is a novel therapy for the glycosphingolipidoses. We report the use of SRT in a patient with NP-C. We show that depletion of glycosphingolipids by miglustat treatment reduces pathological lipid storage, improves endosomal uptake and normalises lipid trafficking in peripheral blood B lymphocytes. The demonstration that treatment with miglustat, which has no direct effect on cholesterol metabolism, corrects the abnormal lipid trafficking seen in B lymphocytes in NP-C indicates that glycosphingolipid accumulation is the primary pathogenetic event in NP-C. These observations support the use of SRT in patients with this devastating neurodegenerative disease.
    Neurobiology of Disease 09/2004; 16(3):654-8. DOI:10.1016/j.nbd.2004.05.002 · 5.20 Impact Factor
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    ABSTRACT: The imino sugar N-butyldeoxynojirimycin (NB-DNJ) is a glucose analogue which inhibits the glycoprotein N-glycan processing enzymes alpha-glucosidases I and II and the ceramide glucosyltransferase that catalyses the first step of glycosphingolipid biosynthesis. This and other N-alkylated DNJ compounds have the potential to inhibit other glucosidase, including acid alpha-glucosidase and alpha-1,6-glucosidase, enzymes involved in glycogen breakdown. We have investigated the effect of NB-DNJ and N-nonyldeoxynojirimycin (NN-DNJ) on glycogen catabolism. Both NB-DNJ and NN-DNJ were potent inhibitors of acid alpha-glucosidase and alpha-1,6-glucosidase in vitro. NB-DNJ and NN-DNJ inhibited liver glycogen breakdown in vivo in fasting mice. Inhibition of glycogen catabolism occurred in the cytosol and lysosomes. The liver glycogen breakdown inhibition was only induced at high doses of NB-DNJ, whereas NN-DNJ caused glycogen accumulation at lower doses. The in vivo effect of NB-DNJ on liver glycogen was transient as there was no inhibition of breakdown after 90 days of treatment. The inhibition by NN-DNJ, was more pronounced, reached a plateau at 50 days and then remained unchanged. Increased glycogen was also observed in skeletal muscle in NB-DNJ- and NN-DNJ-treated mice. Since the effects on glycogen metabolism by NB-DNJ are transient and only occur at high concentrations, it is not predicted that glycogen breakdown will be impaired in patients receiving NB-DNJ therapy. NN-DNJ is the prototype of long alkyl chain derivatives of DNJ that are entering pre-clinical development as potential hepatitis B/hepatitis C (HBV/HCV) therapeutics. Depending on the dose of these compounds used, there is the potential for glycogen catabolism to be partially impaired in experimental animals and man.
    Biochemical Pharmacology 03/2004; 67(4):697-705. DOI:10.1016/j.bcp.2003.09.036 · 4.65 Impact Factor
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    ABSTRACT: Mouse models of the G(M2) gangliosidoses, Tay-Sachs and Sandhoff disease, are null for the hexosaminidase alpha and beta subunits respectively. The Sandhoff (Hexb-/-) mouse has severe neurological disease and mimics the human infantile onset variant. However, the Tay-Sachs (Hexa-/-) mouse model lacks an overt phenotype as mice can partially bypass the blocked catabolic pathway and escape disease. We have investigated whether a subset of Tay-Sachs mice develop late onset disease. We have found that approximately 65% of the mice develop one or more clinical signs of the disease within their natural life span (n = 52, P < 0.0001). However, 100% of female mice with repeat breeding histories developed late onset disease at an earlier age (n = 21, P < 0.0001) and displayed all clinical features. Repeat breeding of a large cohort of female Tay-Sachs mice confirmed that pregnancy induces late onset Tay-Sachs disease. Onset of symptoms correlated with reduced up-regulation of hexosaminidase B, a component of the bypass pathway.
    Neurobiology of Disease 08/2002; 10(3):201-10. DOI:10.1006/nbdi.2002.0511 · 5.20 Impact Factor
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    ABSTRACT: The functional importance of glycolipids has emphasized the need for more sensitive methods of detection, characterization, and quantification than has often been possible using traditional thin-layer chromatographic techniques. We describe the use of ceramide glycanase and HPLC to identify and quantify gangliosides in which the carbohydrate is in Glcbeta1--> linkage with ceramide. Detection of released carbohydrate was by fluorescent labeling with 2-aminobenzamide at the reducing terminal prior to HPLC analysis. Under the conditions described, ceramide glycanase hydrolyzed all of the common gangliosides studied, offering a broad spectrum of specificity. Release and detection of carbohydrate were linear over a wide range (over two orders of magnitude) of micromolar glycolipid substrate concentrations. Use of an N-linked glycan as an internal standard allowed accurate quantification and a recovery of 93% was achieved. The method additionally maintained the sensitivity (chromatographic peaks containing 1 pmol were readily detected from tissue samples) and comparable resolution to related assays. This was shown by the separation, not only of isomeric carbohydrates from the "a" and "b" series, but also of ganglioside carbohydrate differing only by the presence of either N-acetyl- or N-glycolylneuraminic acid. Application of the method to neutral glycosphingolipids and to tissue samples, including 10-microl quantities of plasma, is illustrated. Glycan structures were confirmed by exoglycosidase digestion and/or matrix-assisted laser desorption/ionization mass spectrometry.
    Analytical Biochemistry 11/2001; 298(2):207-17. DOI:10.1006/abio.2001.5393 · 2.31 Impact Factor
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    F M Platt, G Reinkensmeier, RA Dwek, T D Butters
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    ABSTRACT: The imino sugar N-butyldeoxynojirimycin is an inhibitor of the ceramide-specific glucosyltransferase that catalyzes the first step in glycosphingolipid biosynthesis. It results in extensive glycosphingolipid depletion in cells treated in vitro, without causing toxicity. However, we currently do not know the degree to which glycosphingolipids can be depleted in vivo in a mammalian species. We have therefore administered N-butyldeoxynojirimycin long term to young mice and have found that glycosphingolipid levels are reduced (50-70%) in all tissues examined, without resulting in any overt pathology. When the lymphoid tissues from these mice were examined, they were found to be 50% acellular relative to non-lymphoid tissues. These data implicate a role for glycosphingolipids in the biology of the immune system or indicate an additional as yet unknown activity of N-butyldeoxynojirimycin. Extensive glycosphingolipid depletion resulting from N-butyldeoxynojirimycin administration is therefore well tolerated in adult mice, and this compound may be in an invaluable tool for probing glycosphingolipid functions in vivo. In addition, this drug may be effective in clinical situations where glycosphingolipid depletion would be desirable, such as the in the treatment of the human glycosphingolipidoses.
    Journal of Biological Chemistry 09/1997; 272(31):19365-72. DOI:10.1074/jbc.272.31.19365 · 4.60 Impact Factor
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    ABSTRACT: Glucosylated oligomannose N-linked oligosaccharides (Glc(x)Man9GlcNAc2 where x = 1-3) are not normally found on mature glycoproteins but are involved in the early stages of glycoprotein biosynthesis and folding as (i) recognition elements during protein N-glycosylation and chaperone recognition and (ii) substrates in the initial steps of N-glycan processing. By inhibiting the first steps of glycan processing in CHO cells using the alpha-glucosidase inhibitor N-butyl-deoxynojirimycin, we have produced sufficient Glc3Man7GlcNAc2 for structural analysis by nuclear magnetic resonance (NMR) spectroscopy. Our results show the glucosyl cap to have a single, well-defined conformation independent of the rest of the saccharide. Comparison with the conformation of Man9GlcNAc2, previously determined by NMR and molecular dynamics, shows the mannose residues to be largely unaffected by the presence of the glucosyl cap. Sequential enzymatic cleavage of the glucose residues does not affect the conformation of the remaining saccharide. Modelling of the Glc3Man9GlcNAc2, Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2 conformations shows the glucose residues to be fully accessible for recognition. A more detailed analysis of the conformations allows potential recognition epitopes on the glycans to be identified and can form the basis for understanding the specificity of the glucosidases and chaperones (such as calnexin) that recognize these glycans, with implications for their mechanisms of action.
    The EMBO Journal 08/1997; 16(14):4302-10. · 10.75 Impact Factor
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    ABSTRACT: The glycosphingolipid (GSL) lysosomal storage diseases result from the inheritance of defects in the genes encoding the enzymes required for catabolism of GSLs within lysosomes. A strategy for the treatment of these diseases, based on an inhibitor of GSL biosynthesis N-butyldeoxynojirimycin, was evaluated in a mouse model of Tay-Sachs disease. When Tay-Sachs mice were treated with N-butyldeoxynojirimycin, the accumulation of GM2 in the brain was prevented, with the number of storage neurons and the quantity of ganglioside stored per cell markedly reduced. Thus, limiting the biosynthesis of the substrate (GM2) for the defective enzyme (beta-hexosaminidase A) prevents GSL accumulation and the neuropathology associated with its lysosomal storage.
    Science 05/1997; 276(5311):428-31. · 31.48 Impact Factor

Publication Stats

670 Citations
106.41 Total Impact Points

Institutions

  • 1997–2011
    • University of Oxford
      • Department of Biochemistry
      Oxford, England, United Kingdom
  • 2004
    • University of Cambridge
      • Department of Medicine
      Cambridge, ENG, United Kingdom