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1.  Bisubstrate UDP–peptide conjugates as human O-GlcNAc transferase inhibitors 
Biochemical Journal  2014;457(Pt 3):497-502.
Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C3 linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates.
Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, inhibiting glycosyltransfer on to protein and peptide substrates.
doi:10.1042/BJ20131272
PMCID: PMC3927924  PMID: 24256146
bisubstrate analogue inhibitor; glycosyltransferase; O-GlcNAc; rational drug design; DIPEA, N,N-di-isopropylethylamine; DMF, dimethylformamide; goblin, OGT bisubstrate-linked inhibitor; h, human; HRMS, high-resolution MS; MP, p-methoxyphenyl; OGA, O-GlcNAc hydrolase; OGT, O-GlcNAc:polypeptidyl transferase; TAB1, TGF (transforming growth factor)-β-activated kinase-binding protein 1
2.  O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis 
Nature chemical biology  2012;8(12):969-974.
Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-GlcNAc transferase using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human O-GlcNAc transferase recognizes the sugar donor and acceptor peptide and employs a novel catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base, as well as an essential lysine. This mechanism appears to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate, and explains the unexpected specificity of a recently reported metabolic O-GlcNAc transferase inhibitor.
doi:10.1038/nchembio.1108
PMCID: PMC3509171  PMID: 23103942
3.  Probing Elongating and Branching β-d-Galactosyltransferase Activities in Leishmania Parasites by Making Use of Synthetic Phosphoglycans 
ACS chemical biology  2011;6(6):648-657.
Protozoan parasites of the genus Leishmania synthesize lipophosphoglycans (LPGs), phosphoglycans and proteophosphoglycans that contain phosphosaccharide repeat units of [−6)Gal(β1-4)Man(α1-OPO3H−]. The repeat structures are assembled by sequential addition of Manα1-OPO3H and β-Gal. In this study, an UDP-Gal-dependent activity was detected in L. donovani and L. major membranes using synthetic phospho-oligosaccharide fragments of lipophosphoglycan as acceptor substrates. Incubation of a microsomal preparation from L. donovani or L. major parasites with synthetic substrates and UDP-[6-3H]Gal resulted in incorporation of radiolabel into these exogenous acceptors. The [3H]galactose-labeled products were characterized by degradation into radioactive, low molecular mass fragments upon hydrolysis with mild acid and treatment with β-galactosidases. We showed that the activity detected with L. donovani membranes is the elongating β-d-galactosyltransferase associated with LPG phosphosaccharide backbone biosynthesis (eGalT). The eGalT activity showed a requirement for the presence of at least one phosphodiester group in the substrate and it was enhanced dramatically when two or three phosphodiester groups were present. Using the same substrates we detected two types of galactosyltransferase activity in L. major membranes: the elongating β-d-galactosyltransferase and a branching β-d-galactosyltransferase (bGalT). Both L. major enzymes required a minimum of one phosphodiester group present in the substrate, but acceptors with two or three phosphodiester groups were found to be superior.
doi:10.1021/cb100416j
PMCID: PMC3659391  PMID: 21425873
4.  Synergy of Peptide and Sugar in O-GlcNAcase Substrate Recognition 
Chemistry & Biology  2012;19(2):173-178.
Summary
Protein O-GlcNAcylation is an essential reversible posttranslational modification in higher eukaryotes. O-GlcNAc addition and removal is catalyzed by O-GlcNAc transferase and O-GlcNAcase, respectively. We report the molecular details of the interaction of a bacterial O-GlcNAcase homolog with three different synthetic glycopeptides derived from characterized O-GlcNAc sites in the human proteome. Strikingly, the peptides bind a conserved O-GlcNAcase substrate binding groove with similar orientation and conformation. In addition to extensive contacts with the sugar, O-GlcNAcase recognizes the peptide backbone through hydrophobic interactions and intramolecular hydrogen bonds, while avoiding interactions with the glycopeptide side chains. These findings elucidate the molecular basis of O-GlcNAcase substrate specificity, explaining how a single enzyme achieves cycling of the complete O-GlcNAc proteome. In addition, this work will aid development of O-GlcNAcase inhibitors that target the peptide binding site.
Highlights
► Multiple O-GlcNAc peptides bind OGA with similar orientation and conformations ► OGA interacts with the peptide backbone of substrates, not with side chains ► Intramolecular hydrogen bonds affect substrate conformation and Km of glycopeptides ► Different OGA inhibitors display varying levels of peptide mimicry
doi:10.1016/j.chembiol.2012.01.011
PMCID: PMC3476531  PMID: 22365600
5.  O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release 
The EMBO Journal  2012;31(6):1394-1404.
O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release
The protein kinase TAK1 plays an important role in pro-inflammatory cytokine signalling. Interleukin-1- and osmotic stress-induced O-GlcNAcylation of its regulatory subunit TAB1 is required for full TAK1 activation to induce downstream cytokine production, linking this protein modification to innate immunity signalling.
Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is a key serine/threonine protein kinase that mediates signals transduced by pro-inflammatory cytokines such as transforming growth factor-β, tumour necrosis factor (TNF), interleukin-1 (IL-1) and wnt family ligands. TAK1 is found in complex with binding partners TAB1–3, phosphorylation and ubiquitination of which has been found to regulate TAK1 activity. In this study, we show that TAB1 is modified with N-acetylglucosamine (O-GlcNAc) on a single site, Ser395. With the help of a novel O-GlcNAc site-specific antibody, we demonstrate that O-GlcNAcylation of TAB1 is induced by IL-1 and osmotic stress, known inducers of the TAK1 signalling cascade. By reintroducing wild-type or an O-GlcNAc-deficient mutant TAB1 (S395A) into Tab1−/− mouse embryonic fibroblasts, we determined that O-GlcNAcylation of TAB1 is required for full TAK1 activation upon stimulation with IL-1/osmotic stress, for downstream activation of nuclear factor κB and finally production of IL-6 and TNFα. This is one of the first examples of a single O-GlcNAc site on a signalling protein modulating a key innate immunity signalling pathway.
doi:10.1038/emboj.2012.8
PMCID: PMC3321193  PMID: 22307082
cytokine; glycobiology; innate immunity; O-GlcNAc; signal transduction
6.  Cell-Penetrant, Nanomolar O-GlcNAcase Inhibitors Selective against Lysosomal Hexosaminidases 
Chemistry & Biology  2010;17(11):1250-1255.
Summary
Posttranslational modification of metazoan nucleocytoplasmic proteins with N-acetylglucosamine (O-GlcNAc) is essential, dynamic, and inducible and can compete with protein phosphorylation in signal transduction. Inhibitors of O-GlcNAcase, the enzyme removing O-GlcNAc, are useful tools for studying the role of O-GlcNAc in a range of cellular processes. We report the discovery of nanomolar OGA inhibitors that are up to 900,000-fold selective over the related lysosomal hexosaminidases. When applied at nanomolar concentrations on live cells, these cell-penetrant molecules shift the O-GlcNAc equilibrium toward hyper-O-GlcNAcylation with EC50 values down to 3 nM and are thus invaluable tools for the study of O-GlcNAc cell biology.
Highlights
► Structure-guided design of human O-GlcNAcase inhibitors, GlcNAcstatins ► The GlcNAcstatins are competitive, nanomolar inhibitors ► The molecular basis of the exquisite selectivity revealed by crystallography ► First direct evidence of O-GlcNAcase inhibitors penetrating cells
doi:10.1016/j.chembiol.2010.09.014
PMCID: PMC3032886  PMID: 21095575
7.  Human OGA binds substrates in a conserved peptide recognition groove 
Biochemical Journal  2010;432(Pt 1):1-7.
Modification of cellular proteins with O-GlcNAc (O-linked N-acetylglucosamine) competes with protein phosphorylation and regulates a plethora of cellular processes. O-GlcNAcylation is orchestrated by two opposing enzymes, O-GlcNAc transferase and OGA (O-GlcNAcase or β-N-acetylglucosaminidase), which recognize their target proteins via as yet unidentified mechanisms. In the present study, we uncovered the first insights into the mechanism of substrate recognition by human OGA. The structure of a novel bacterial OGA orthologue reveals a putative substrate-binding groove, conserved in metazoan OGAs. Guided by this structure, conserved amino acids lining this groove in human OGA were mutated and the activity on three different substrate proteins [TAB1 (transforming growth factor-β-activated protein kinase 1-binding protein 1), FoxO1 (forkhead box O1) and CREB (cAMP-response-element-binding protein)] was tested in an in vitro deglycosylation assay. The results provide the first evidence that human OGA may possess a substrate-recognition mechanism that involves interactions with O-GlcNAcylated proteins beyond the GlcNAc-binding site, with possible implications for differential regulation of cycling of O-GlcNAc on different proteins.
doi:10.1042/BJ20101338
PMCID: PMC2973230  PMID: 20863279
β-N-acetylglucosaminidase; O-linked N-acetylglucosamine (O-GlcNAc); peptide recognition groove; protein glycosylation; CpNagJ, Clostridium perfringens NagJ; CREB, cAMP-response-element-binding protein; Fmoc, fluoren-9-ylmethoxycarbonyl; FoxO1, forkhead box O1; GST, glutathione transferase; HAT, histone acetyltransferase; HEK, human embryonic kidney; LC, liquid chromatography; 4MU-GlcNAc, 4-methylumbelliferyl-β-N-acetylglucosamine; OGA, O-GlcNAcase or β-N-acetylglucosaminidase; hOGA, human OGA; OgOGA, Oceanicola granulosus OGA; OGT, O-GlcNAc transferase; O-GlcNAc, O-linked N-acetylglucosamine; pNP-GlcNAc, p-nitrophenyl-β-N-acetylglucosamine; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate; RMSD, root mean square deviation; TAB1, transforming growth factor-β-activated protein kinase 1-binding protein 1
8.  An efficient and versatile synthesis of GlcNAcstatins—potent and selective O-GlcNAcase inhibitors built on the tetrahydroimidazo[1,2-a]pyridine scaffold 
Tetrahedron  2010;66(39-3):7838-7849.
We report a novel approach to the synthesis of GlcNAcstatins—members of an emerging family of potent and selective inhibitors of peptidyl O-GlcNAc hydrolase build upon tetrahydroimidazo[1,2-a]pyridine scaffold. Making use of a streamlined synthetic sequence featuring de novo synthesis of imidazoles from glyoxal, ammonia and aldehydes, a properly functionalised linear GlcNAcstatin precursor has been efficiently prepared starting from methyl 3,4-O-(2′,3′-dimethoxybutane-2′,3′-diyl)-α-d-mannopyranoside. Subsequent ring closure of the linear precursor in an intramolecular SN2 process furnished the key fused d-mannose-imidazole GlcNAcstatin precursor in excellent yield. Finally, a sequence of transformations of this key intermediate granted expeditious access to a variety of the target compounds bearing a C(2)-phenethyl group and a range of N(8) acyl substituents. The versatility of the new approach stems from an appropriate choice of a set of acid labile permanent protecting groups on the monosaccharide starting material. Application was demonstrated by the synthesis of GlcNAcstatins containing polyunsaturated and thiol-containing amido substituents.
Graphical abstract
doi:10.1016/j.tet.2010.07.037
PMCID: PMC2956484  PMID: 20976183
9.  Substrate and product analogues as human O-GlcNAc transferase inhibitors 
Amino Acids  2010;40(3):781-792.
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.
Electronic supplementary material
The online version of this article (doi:10.1007/s00726-010-0688-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s00726-010-0688-y
PMCID: PMC3040809  PMID: 20640461
O-GlcNAc; Post-translational modification; Inhibitor; Signalling; Crystal structure
10.  GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation 
Biochemical Journal  2009;420(Pt 2):221-227.
O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.
doi:10.1042/BJ20090110
PMCID: PMC2691177  PMID: 19275764
GlcNAcstatin; inhibition; O-GlcNAc; O-GlcNAcase; GST, glutathione transferase; HEK, human embryonic kidney; Hex, hexosaminidase; 4MU-NAG, 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide; OGA, O-GlcNAcase; CpOGA, Clostridium perfringens OGA; hOGA, human OGA; OGT, O-GlcNAc transferase; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenylcarbamate
11.  Structural insights into mechanism and specificity of O-GlcNAc transferase 
The EMBO Journal  2008;27(20):2780-2788.
Post-translational modification of protein serines/threonines with N-acetylglucosamine (O-GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O-GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X-ray crystallography and mutagenesis, that OGT adopts the (metal-independent) GT-B fold and binds a UDP-GlcNAc analogue at the bottom of a highly conserved putative peptide-binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 Å putative interaction surface, whereas the previously predicted phosphatidylinositide-binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.
doi:10.1038/emboj.2008.186
PMCID: PMC2556091  PMID: 18818698
glycobiology; O-GlcNAc; protein structure; signal transduction
12.  Chemical Dissection of the Link between Streptozotocin, O-GlcNAc, and Pancreatic Cell Death 
Chemistry & Biology  2008;15(8):799-807.
Summary
Streptozotocin is a natural product that selectively kills insulin-secreting β cells, and is widely used to generate mouse models of diabetes or treat pancreatic tumors. Several studies suggest that streptozotocin toxicity stems from its N-nitrosourea moiety releasing nitric oxide and possessing DNA alkylating activity. However, it has also been proposed that streptozotocin induces apoptosis by inhibiting O-GlcNAcase, an enzyme that, together with O-GlcNAc transferase, is important for dynamic intracellular protein O-glycosylation. We have used galacto-streptozotocin to chemically dissect the link between O-GlcNAcase inhibition and apoptosis. Using X-ray crystallography, enzymology, and cell biological studies on an insulinoma cell line, we show that, whereas streptozotocin competitively inhibits O-GlcNAcase and induces apoptosis, its galacto-configured derivative no longer inhibits O-GlcNAcase, yet still induces apoptosis. This supports a general chemical poison mode of action for streptozotocin, suggesting the need for using more specific inhibitors to study protein O-GlcNAcylation.
doi:10.1016/j.chembiol.2008.06.010
PMCID: PMC2568864  PMID: 18721751
CHEMBIO; SIGNALING

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