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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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X-ray crystallographic determination of inhibitor binding modes. a Stereo figure of XcOGT in complex with UDP-S-GlcNAc and superposition of the previously described XcOGT–UDP-C-GlcNAc complex [PDB entry 2JLB (Clarke et al. 2008)]. XcOGT active site residues are shown in sticks with grey carbon, red oxygen, blue nitrogen atoms. UDP-S-GlcNAc and UDP-C-GlcNAc are shown with green and purple carbon, respectively. Hydrogen bonds for the XcOGT–UDP-S-GlcNAc complex are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.75σ) is shown as cyan chickenwire. b Stereo figure of XcOGT in complex with C-UDP compared to the UDP complex [PDB entry 2VSN (Martinez-Fleites et al. 2008)]. XcOGT active site residues are shown in sticks with grey carbon, red oxygen, blue nitrogen atoms (transparent sticks for XcOGT–UDP), ligands shown in green carbon atoms. Active site residues involved in hydrogen bonds are labelled. Hydrogen bonds are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.25σ) is shown as cyan chickenwire. c Stereo figure of XcOGT in complex with alloxan. XcOGT active site are shown in sticks with green carbon, red oxygen, blue nitrogen atoms in the active site of XcOGT (sticks with grey carbon atoms). Hydrogen bonds are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.25σ) is shown as cyan chickenwire. Black dashed lines showing hydrogen bonds for XcOGT–alloxan complex
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Fig2: X-ray crystallographic determination of inhibitor binding modes. a Stereo figure of XcOGT in complex with UDP-S-GlcNAc and superposition of the previously described XcOGT–UDP-C-GlcNAc complex [PDB entry 2JLB (Clarke et al. 2008)]. XcOGT active site residues are shown in sticks with grey carbon, red oxygen, blue nitrogen atoms. UDP-S-GlcNAc and UDP-C-GlcNAc are shown with green and purple carbon, respectively. Hydrogen bonds for the XcOGT–UDP-S-GlcNAc complex are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.75σ) is shown as cyan chickenwire. b Stereo figure of XcOGT in complex with C-UDP compared to the UDP complex [PDB entry 2VSN (Martinez-Fleites et al. 2008)]. XcOGT active site residues are shown in sticks with grey carbon, red oxygen, blue nitrogen atoms (transparent sticks for XcOGT–UDP), ligands shown in green carbon atoms. Active site residues involved in hydrogen bonds are labelled. Hydrogen bonds are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.25σ) is shown as cyan chickenwire. c Stereo figure of XcOGT in complex with alloxan. XcOGT active site are shown in sticks with green carbon, red oxygen, blue nitrogen atoms in the active site of XcOGT (sticks with grey carbon atoms). Hydrogen bonds are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.25σ) is shown as cyan chickenwire. Black dashed lines showing hydrogen bonds for XcOGT–alloxan complex

Mentions: To investigate the binding mode of the UDP-S-GlcNAc substrate analogue, XcOGT protein crystals were soaked with the compound. Collection of synchrotron diffraction data and subsequent refinement yielded a final model with statistics shown in Table 3. UDP-S-GlcNAc binds in the active site of XcOGT (Fig. 2a). The uracil moiety occupies a pocket formed by Lys441 to His444, whilst stacking with the side chain of Tyr447 (Fig. 2a). The uracil moiety is involved in a total of three hydrogen bonds with the protein, with the backbone of Leu442/Pro443 and the His444 side chain (Fig. 2a). The ribose moiety of UDP-S-GlcNAc makes a hydrogen bond with Asp471 (Fig. 2), a side chain that is conserved in OGTs from organisms across the evolutionary spectrum. Recent studies reported that a mutant of the equivalent position in hOGT (Asp925) is no longer able to O-GlcNAcylate an acceptor substrate (Clarke et al. 2008; Martinez-Fleites et al. 2008). SPR measurements show that the D471A-XcOGT mutant no longer binds UDP–GlcNAc (Table 2).Table 3


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)

X-ray crystallographic determination of inhibitor binding modes. a Stereo figure of XcOGT in complex with UDP-S-GlcNAc and superposition of the previously described XcOGT–UDP-C-GlcNAc complex [PDB entry 2JLB (Clarke et al. 2008)]. XcOGT active site residues are shown in sticks with grey carbon, red oxygen, blue nitrogen atoms. UDP-S-GlcNAc and UDP-C-GlcNAc are shown with green and purple carbon, respectively. Hydrogen bonds for the XcOGT–UDP-S-GlcNAc complex are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.75σ) is shown as cyan chickenwire. b Stereo figure of XcOGT in complex with C-UDP compared to the UDP complex [PDB entry 2VSN (Martinez-Fleites et al. 2008)]. XcOGT active site residues are shown in sticks with grey carbon, red oxygen, blue nitrogen atoms (transparent sticks for XcOGT–UDP), ligands shown in green carbon atoms. Active site residues involved in hydrogen bonds are labelled. Hydrogen bonds are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.25σ) is shown as cyan chickenwire. c Stereo figure of XcOGT in complex with alloxan. XcOGT active site are shown in sticks with green carbon, red oxygen, blue nitrogen atoms in the active site of XcOGT (sticks with grey carbon atoms). Hydrogen bonds are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.25σ) is shown as cyan chickenwire. Black dashed lines showing hydrogen bonds for XcOGT–alloxan complex
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Fig2: X-ray crystallographic determination of inhibitor binding modes. a Stereo figure of XcOGT in complex with UDP-S-GlcNAc and superposition of the previously described XcOGT–UDP-C-GlcNAc complex [PDB entry 2JLB (Clarke et al. 2008)]. XcOGT active site residues are shown in sticks with grey carbon, red oxygen, blue nitrogen atoms. UDP-S-GlcNAc and UDP-C-GlcNAc are shown with green and purple carbon, respectively. Hydrogen bonds for the XcOGT–UDP-S-GlcNAc complex are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.75σ) is shown as cyan chickenwire. b Stereo figure of XcOGT in complex with C-UDP compared to the UDP complex [PDB entry 2VSN (Martinez-Fleites et al. 2008)]. XcOGT active site residues are shown in sticks with grey carbon, red oxygen, blue nitrogen atoms (transparent sticks for XcOGT–UDP), ligands shown in green carbon atoms. Active site residues involved in hydrogen bonds are labelled. Hydrogen bonds are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.25σ) is shown as cyan chickenwire. c Stereo figure of XcOGT in complex with alloxan. XcOGT active site are shown in sticks with green carbon, red oxygen, blue nitrogen atoms in the active site of XcOGT (sticks with grey carbon atoms). Hydrogen bonds are indicated by black dashed lines. Unbiased /Fo/−/Fc/, ϕcalc electron density map (2.25σ) is shown as cyan chickenwire. Black dashed lines showing hydrogen bonds for XcOGT–alloxan complex
Mentions: To investigate the binding mode of the UDP-S-GlcNAc substrate analogue, XcOGT protein crystals were soaked with the compound. Collection of synchrotron diffraction data and subsequent refinement yielded a final model with statistics shown in Table 3. UDP-S-GlcNAc binds in the active site of XcOGT (Fig. 2a). The uracil moiety occupies a pocket formed by Lys441 to His444, whilst stacking with the side chain of Tyr447 (Fig. 2a). The uracil moiety is involved in a total of three hydrogen bonds with the protein, with the backbone of Leu442/Pro443 and the His444 side chain (Fig. 2a). The ribose moiety of UDP-S-GlcNAc makes a hydrogen bond with Asp471 (Fig. 2), a side chain that is conserved in OGTs from organisms across the evolutionary spectrum. Recent studies reported that a mutant of the equivalent position in hOGT (Asp925) is no longer able to O-GlcNAcylate an acceptor substrate (Clarke et al. 2008; Martinez-Fleites et al. 2008). SPR measurements show that the D471A-XcOGT mutant no longer binds UDP–GlcNAc (Table 2).Table 3

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