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Substrate and product analogues as human O-GlcNAc transferase inhibitors.

Dorfmueller HC, Borodkin VS, Blair DE, Pathak S, Navratilova I, van Aalten DM - Amino Acids (2010)

Bottom Line: Specific inhibitors of human OGT would be useful tools to probe the role of this post-translational modification in regulating processes in the living cell.Here, we describe the synthesis of novel UDP-GlcNAc/UDP analogues and evaluate their inhibitory properties and structural binding modes in vitro alongside alloxan, a previously reported weak OGT inhibitor.While the novel analogues are not active on living cells, they inhibit the enzyme in the micromolar range and together with the structural data provide useful templates for further optimisation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, Scotland, UK.

ABSTRACT
Protein glycosylation on serine/threonine residues with N-acetylglucosamine (O-GlcNAc) is a dynamic, inducible and abundant post-translational modification. It is thought to regulate many cellular processes and there are examples of interplay between O-GlcNAc and protein phosphorylation. In metazoa, a single, highly conserved and essential gene encodes the O-GlcNAc transferase (OGT) that transfers GlcNAc onto substrate proteins using UDP-GlcNAc as the sugar donor. Specific inhibitors of human OGT would be useful tools to probe the role of this post-translational modification in regulating processes in the living cell. Here, we describe the synthesis of novel UDP-GlcNAc/UDP analogues and evaluate their inhibitory properties and structural binding modes in vitro alongside alloxan, a previously reported weak OGT inhibitor. While the novel analogues are not active on living cells, they inhibit the enzyme in the micromolar range and together with the structural data provide useful templates for further optimisation.

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Design, synthesis and inhibitory properties of hOGT substrate/product analogues. a Chemical structures of UDP–GlcNAc, the substrate of hOGT, the substrate analogues UDP-C-GlcNAc and UDP-S-GlcNAc, the hOGT product UDP and the analogue C-UDP and alloxan. The atoms in the uracil moiety are numbered. Alloxan ring atoms are numbered according to the uracil numbering system. b Dose–response curves of the inhibitors with hOGT. The transfer of GlcNAc from UDP–GlcNAc onto a peptide [DEBtide (Blair et al. manuscript submitted)] by hOGT was assayed in presence of increasing concentrations of UDP-S-GlcNAc, UDP-C-GlcNAc, alloxan and C-UDP. The remaining activity is plotted against the inhibitor concentration. Data were fitted using the standard IC50 equation in the GraFit program (Leatherbarrow 2001). c Reagents and conditions for UDP-S-GlcNAc synthesis: (i) Bis(9H-fluoren-9-ylmethyl)-diisopropylamidophosphite, 1H-tetrazole, MeCN, RT, 30 min; then dimethyldioxirane, −40°C to −20°C, 30 min; (ii) DCM:Et3N (4:1), RT, 24 h; (iii) (a) 2a + 3, MeCN, RT, 16 h; (b) MeOH:H2O:Et3N (5:2:1), RT, 16 h; followed by size exclusion chromatography
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Fig1: Design, synthesis and inhibitory properties of hOGT substrate/product analogues. a Chemical structures of UDP–GlcNAc, the substrate of hOGT, the substrate analogues UDP-C-GlcNAc and UDP-S-GlcNAc, the hOGT product UDP and the analogue C-UDP and alloxan. The atoms in the uracil moiety are numbered. Alloxan ring atoms are numbered according to the uracil numbering system. b Dose–response curves of the inhibitors with hOGT. The transfer of GlcNAc from UDP–GlcNAc onto a peptide [DEBtide (Blair et al. manuscript submitted)] by hOGT was assayed in presence of increasing concentrations of UDP-S-GlcNAc, UDP-C-GlcNAc, alloxan and C-UDP. The remaining activity is plotted against the inhibitor concentration. Data were fitted using the standard IC50 equation in the GraFit program (Leatherbarrow 2001). c Reagents and conditions for UDP-S-GlcNAc synthesis: (i) Bis(9H-fluoren-9-ylmethyl)-diisopropylamidophosphite, 1H-tetrazole, MeCN, RT, 30 min; then dimethyldioxirane, −40°C to −20°C, 30 min; (ii) DCM:Et3N (4:1), RT, 24 h; (iii) (a) 2a + 3, MeCN, RT, 16 h; (b) MeOH:H2O:Et3N (5:2:1), RT, 16 h; followed by size exclusion chromatography

Mentions: hOGT assays were carried out at room temperature (RT) in 384-well white optiplates (Perkin Elmer). Each assay was performed in a 20 μl reaction volume containing 50 mM Tris buffer, pH 7.5, 2 mM dithiothreitol (DTT), 3 mM MgCl2, 0.05 mg/ml BSA (Thermo), 0.125 μM [3H]UDP–GlcNAc (0.048 Ci mmol−1) (Sigma/ARC), 1 μM biotinylated DEBtide (Blair et al. manuscript submitted) (GlycoBioChem), 50 nM hOGT and various concentrations of test compounds. Test compounds were placed in the columns 1 and 13 of a 384-well polypropylene plate and then serially diluted in 100% DMSO through half log increments using a JANUS 8-channel Varispan automated workstation (PerkinElmer) to give the compound source plate (100× final assay concentration). From this, 0.25 μl of the compounds were transferred to all wells using a Cartesian Hummingbird (Genomics Solution) before 10 μl of either enzyme/peptide mix or peptide alone (control) was added to the assay plates. The reaction was initiated with 10 μl of UDP–GlcNAc and stopped after 150 min with 40 μl of a stop solution containing 0.2 M phosphoric acid, pH 4.0 and 1.5 M MgCl2 and 2.5 mg/ml PVT SPA beads (GE Healthcare). All reaction mixture additions were carried out using a Thermo Scientific WellMate (Matrix). Plates were sealed and read on a TopCount NXT Microplate Scintillation and Luminescence Counter (Perkin Elmer). Dose–response curves were prepared and analysed using GraFit (Leatherbarrow 2001) (Fig. 1b).Fig. 1


Substrate and product analogues as human O-GlcNAc transferase inhibitors.

Dorfmueller HC, Borodkin VS, Blair DE, Pathak S, Navratilova I, van Aalten DM - Amino Acids (2010)

Design, synthesis and inhibitory properties of hOGT substrate/product analogues. a Chemical structures of UDP–GlcNAc, the substrate of hOGT, the substrate analogues UDP-C-GlcNAc and UDP-S-GlcNAc, the hOGT product UDP and the analogue C-UDP and alloxan. The atoms in the uracil moiety are numbered. Alloxan ring atoms are numbered according to the uracil numbering system. b Dose–response curves of the inhibitors with hOGT. The transfer of GlcNAc from UDP–GlcNAc onto a peptide [DEBtide (Blair et al. manuscript submitted)] by hOGT was assayed in presence of increasing concentrations of UDP-S-GlcNAc, UDP-C-GlcNAc, alloxan and C-UDP. The remaining activity is plotted against the inhibitor concentration. Data were fitted using the standard IC50 equation in the GraFit program (Leatherbarrow 2001). c Reagents and conditions for UDP-S-GlcNAc synthesis: (i) Bis(9H-fluoren-9-ylmethyl)-diisopropylamidophosphite, 1H-tetrazole, MeCN, RT, 30 min; then dimethyldioxirane, −40°C to −20°C, 30 min; (ii) DCM:Et3N (4:1), RT, 24 h; (iii) (a) 2a + 3, MeCN, RT, 16 h; (b) MeOH:H2O:Et3N (5:2:1), RT, 16 h; followed by size exclusion chromatography
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Fig1: Design, synthesis and inhibitory properties of hOGT substrate/product analogues. a Chemical structures of UDP–GlcNAc, the substrate of hOGT, the substrate analogues UDP-C-GlcNAc and UDP-S-GlcNAc, the hOGT product UDP and the analogue C-UDP and alloxan. The atoms in the uracil moiety are numbered. Alloxan ring atoms are numbered according to the uracil numbering system. b Dose–response curves of the inhibitors with hOGT. The transfer of GlcNAc from UDP–GlcNAc onto a peptide [DEBtide (Blair et al. manuscript submitted)] by hOGT was assayed in presence of increasing concentrations of UDP-S-GlcNAc, UDP-C-GlcNAc, alloxan and C-UDP. The remaining activity is plotted against the inhibitor concentration. Data were fitted using the standard IC50 equation in the GraFit program (Leatherbarrow 2001). c Reagents and conditions for UDP-S-GlcNAc synthesis: (i) Bis(9H-fluoren-9-ylmethyl)-diisopropylamidophosphite, 1H-tetrazole, MeCN, RT, 30 min; then dimethyldioxirane, −40°C to −20°C, 30 min; (ii) DCM:Et3N (4:1), RT, 24 h; (iii) (a) 2a + 3, MeCN, RT, 16 h; (b) MeOH:H2O:Et3N (5:2:1), RT, 16 h; followed by size exclusion chromatography
Mentions: hOGT assays were carried out at room temperature (RT) in 384-well white optiplates (Perkin Elmer). Each assay was performed in a 20 μl reaction volume containing 50 mM Tris buffer, pH 7.5, 2 mM dithiothreitol (DTT), 3 mM MgCl2, 0.05 mg/ml BSA (Thermo), 0.125 μM [3H]UDP–GlcNAc (0.048 Ci mmol−1) (Sigma/ARC), 1 μM biotinylated DEBtide (Blair et al. manuscript submitted) (GlycoBioChem), 50 nM hOGT and various concentrations of test compounds. Test compounds were placed in the columns 1 and 13 of a 384-well polypropylene plate and then serially diluted in 100% DMSO through half log increments using a JANUS 8-channel Varispan automated workstation (PerkinElmer) to give the compound source plate (100× final assay concentration). From this, 0.25 μl of the compounds were transferred to all wells using a Cartesian Hummingbird (Genomics Solution) before 10 μl of either enzyme/peptide mix or peptide alone (control) was added to the assay plates. The reaction was initiated with 10 μl of UDP–GlcNAc and stopped after 150 min with 40 μl of a stop solution containing 0.2 M phosphoric acid, pH 4.0 and 1.5 M MgCl2 and 2.5 mg/ml PVT SPA beads (GE Healthcare). All reaction mixture additions were carried out using a Thermo Scientific WellMate (Matrix). Plates were sealed and read on a TopCount NXT Microplate Scintillation and Luminescence Counter (Perkin Elmer). Dose–response curves were prepared and analysed using GraFit (Leatherbarrow 2001) (Fig. 1b).Fig. 1

Bottom Line: Specific inhibitors of human OGT would be useful tools to probe the role of this post-translational modification in regulating processes in the living cell.Here, we describe the synthesis of novel UDP-GlcNAc/UDP analogues and evaluate their inhibitory properties and structural binding modes in vitro alongside alloxan, a previously reported weak OGT inhibitor.While the novel analogues are not active on living cells, they inhibit the enzyme in the micromolar range and together with the structural data provide useful templates for further optimisation.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, Scotland, UK.

ABSTRACT
Protein glycosylation on serine/threonine residues with N-acetylglucosamine (O-GlcNAc) is a dynamic, inducible and abundant post-translational modification. It is thought to regulate many cellular processes and there are examples of interplay between O-GlcNAc and protein phosphorylation. In metazoa, a single, highly conserved and essential gene encodes the O-GlcNAc transferase (OGT) that transfers GlcNAc onto substrate proteins using UDP-GlcNAc as the sugar donor. Specific inhibitors of human OGT would be useful tools to probe the role of this post-translational modification in regulating processes in the living cell. Here, we describe the synthesis of novel UDP-GlcNAc/UDP analogues and evaluate their inhibitory properties and structural binding modes in vitro alongside alloxan, a previously reported weak OGT inhibitor. While the novel analogues are not active on living cells, they inhibit the enzyme in the micromolar range and together with the structural data provide useful templates for further optimisation.

Show MeSH
Related in: MedlinePlus