Metabolic Inhibitors of O-GlcNAc Transferase That Act In†Vivo Implicate Decreased O-GlcNAc Levels in Leptin-Mediated Nutrient Sensing.

O-Linked glycosylation of serine and threonine residues of nucleocytoplasmic proteins with N-acetylglucosamine (O-GlcNAc) residues is catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc is conserved within mammals and is implicated in a wide range of physiological processes. Herein, we describe metabolic precursor inhibitors of OGT suitable for use both in cells and in vivo in mice. These 5-thiosugar analogues of N-acetylglucosamine are assimilated through a convergent metabolic pathway, most likely involving N-acetylglucosamine-6-phosphate de-N-acetylase (NAGA), to generate a common OGT inhibitor within cells. We show that of these inhibitors, 2-deoxy-2-N-hexanamide-5-thio-d-glucopyranoside (5SGlcNHex) acts in vivo to induce dose- and time-dependent decreases in O-GlcNAc levels in various tissues. Decreased O-GlcNAc correlates, both in vitro within adipocytes and in vivo within mice, with lower levels of the transcription factor Sp1 and the satiety-inducing hormone leptin, thus revealing a link between decreased O-GlcNAc levels and nutrient sensing in peripheral tissues of mammals.

Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism.

O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the -1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue.

Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-ß-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases.

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-ß-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using ß-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors.

In vivo uptake of ß-amyloid by non-plaque associated microglia.

The role of microglia in ß-amyloid (Aß) deposition or clearance in the Alzheimer's disease (AD) brain remains unclear. Previous in vivo studies have focused primarily on the association of microglia with Aß-positive parenchymal plaques, but have given little consideration to the possible interaction between Aß and non-plaque associated microglia. Further, it is not known if microglia play a direct role in mediating Aß uptake following anti-aggregant treatment. We report here the identification of Aß-positive processes throughout the cortex and hippocampus of TgCRND8 mice expressing the human Swedish (KM670/671NL) and Indiana (V717F) amyloid precursor protein mutations, which localized to ionized calcium binding protein-1-positive resident microglia that were not associated with extracellular plaques. Oral administration of 1-deoxy-1-fluoro-scyllo-inositol, a scyllo-inositol analogue, to TgCRND8 mice improved spatial memory impairments and suppressed amyloid pathology in a dose-dependent manner. Further, treatment with 1-deoxy-1- fluoro-scyllo-inositol significantly increased hippocampal intra-microglial Aß levels without stimulating microglial proliferation or peripheral macrophage recruitment. These results reveal a novel, beneficial role for non-plaque associated microglia in the regulation of cerebral Aß levels in a mouse model of AD.

Inhibition of the pneumococcal virulence factor StrH and molecular insights into N-glycan recognition and hydrolysis.

The complete degradation of N-linked glycans by the pathogenic bacterium Streptococcus pneumoniae is facilitated by the large multimodular cell wall-attached exo-ß-D-N-acetylglucosaminidase StrH. Structural dissection of this virulence factor using X-ray crystallography showed it to have two structurally related glycoside hydrolase family 20 catalytic domains, which displayed the expected specificity for complex N-glycans terminating in N-acetylglucosamine but exhibited unexpected differences in their preferences for the substructures present in these glycans. The structures of the two catalytic domains in complex with unhydrolyzed substrates, including an N-glycan possessing a bisecting N-acetylglucosamine residue, revealed the specific architectural features in the active sites that confer their differential specificities. Inhibitors of StrH are demonstrated to be effective tools in modulating the interaction of StrH with components of the host, such as the innate immune system. Overall, new structural and functional insight into a carbohydrate-mediated component of the pneumococcus-host interaction is provided.

The conformation and function of a multimodular glycogen-degrading pneumococcal virulence factor.

SpuA is a large multimodular cell wall-attached enzyme involved in the degradation of glycogen by the pathogenic bacterium Streptococcus pneumoniae. The deletion of the gene encoding SpuA from the bacterium resulted in a strain with reduced competitiveness in a mouse model of virulence relative to the parent strain, linking the degradation of host-glycogen to the virulence of the bacterium. Through the combined use of X-ray crystallography, small-angle X-ray scattering, and inhibitor binding, the molecular features involved in substrate recognition by this complex protein are revealed. This uniquely illustrates the complexity of the active site, the conformational changes incurred during carbohydrate binding by this protein, and the interaction and cooperation of its composite modules during this process. New insight into the function of this particular pneumococcal virulence factor is provided along with substantial contributions to the nascent framework for understanding the structural and functional interplay between modules in multimodular carbohydrate-active enzymes.

Analysis of a new family of widely distributed metal-independent alpha-mannosidases provides unique insight into the processing of N-linked glycans.

The modification of N-glycans by α-mannosidases is a process that is relevant to a large number of biologically important processes, including infection by microbial pathogens and colonization by microbial symbionts. At present, the described mannosidases specific for α1,6-mannose linkages are very limited in number. Through structural and functional analysis of two sequence-related enzymes, one from Streptococcus pneumoniae (SpGH125) and one from Clostridium perfringens (CpGH125), a new glycoside hydrolase family, GH125, is identified and characterized. Analysis of SpGH125 and CpGH125 reveal them to have exo-α1,6-mannosidase activity consistent with specificity for N-linked glycans having their α1,3-mannose branches removed. The x-ray crystal structures of SpGH125 and CpGH125 obtained in apo-, inhibitor-bound, and substrate-bound forms provide both mechanistic and molecular insight into how these proteins, which adopt an (α/α)(6)-fold, recognize and hydrolyze the α1,6-mannosidic bond by an inverting, metal-independent catalytic mechanism. A phylogenetic analysis of GH125 proteins reveals this to be a relatively large and widespread family found frequently in bacterial pathogens, bacterial human gut symbionts, and a variety of fungi. Based on these studies we predict this family of enzymes will primarily comprise such exo-α1,6-mannosidases.

Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells.

Glycosyltransferases are ubiquitous enzymes that catalyze the assembly of glycoconjugates throughout all kingdoms of nature. A long-standing problem is the rational design of probes that can be used to manipulate glycosyltransferase activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analog that inhibits, with high potency both in vitro and in cells, the human glycosyltransferase responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show that the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analog. Treatment of cells with the precursor lowers O-GlcNAc in a targeted manner with a single-digit micromolar EC(50). This approach to inhibition of glycosyltransferases should be applicable to other members of this superfamily of enzymes and enable their manipulation in a biological setting.

NagZ inactivation prevents and reverts beta-lactam resistance, driven by AmpD and PBP 4 mutations, in Pseudomonas aeruginosa.

AmpC hyperproduction is the most frequent mechanism of resistance to penicillins and cephalosporins in Pseudomonas aeruginosa and is driven by ampD mutations or the recently described inactivation of dacB, which encodes the nonessential penicillin-binding protein (PBP) PBP 4. Recent work showed that nagZ inactivation attenuates beta-lactam resistance in ampD mutants. Here we explored whether the same could be true for the dacB mutants with dacB mutations alone or in combination with ampD mutations. The inactivation of nagZ restored the wild-type beta-lactam MICs and ampC expression of PAO1 dacB and ampD mutants and dramatically reduced the MICs (for example, the MIC for ceftazidime dropped from 96 to 4 microg/ml) and the level of ampC expression (from ca. 1,000-fold to ca. 50-fold higher than that for PAO1) in the dacB-ampD double mutant. On the other hand, nagZ inactivation had little effect on the inducibility of AmpC. The NagZ inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate attenuated the beta-lactam resistance of the AmpC-hyperproducing strains, showing a greater effect on the dacB mutant (reducing the ceftazidime MICs from 24 to 6 microg/ml) than the ampD mutant (reducing the MICs from 8 to 4 microg/ml). Additionally, nagZ inactivation in the dacB mutant blocked the overexpression of creD (blrD), which is a marker of the activation of the CreBC (BlrAB) regulator involved in the resistance phenotype. Finally, through population analysis, we show that the inactivation of nagZ dramatically reduces the capacity of P. aeruginosa to develop ceftazidime resistance, since spontaneous mutants were not obtained at concentrations > or = 8 microg/ml (the susceptibility breakpoint) for the nagZ mutant but were obtained with wild-type PAO1. Therefore, NagZ is envisaged to be a candidate target for preventing and reverting beta-lactam resistance in P. aeruginosa.

Small molecule beta-amyloid inhibitors that stabilize protofibrillar structures in vitro improve cognition and pathology in a mouse model of Alzheimer's disease.

Beta-amyloid (Abeta) peptides are thought to play a major role in the pathogenesis of Alzheimer's disease. Compounds that disrupt the kinetic pathways of Abeta aggregation may be useful in elucidating the role of oligomeric, protofibrillar and fibrillar Abeta in the etiology of the disease. We have previously reported that scyllo-inositol inhibits Abeta(42) fibril formation but the mechanism(s) by which this occurs has not been investigated in detail. Using a series of scyllo-inositol derivatives in which one or two hydroxyl groups were replaced with hydrogen, chlorine or methoxy substituents, we examined the role of hydrogen bonding and hydrophobicity in the structure-function relationship of scyllo-inositol-Abeta binding. We report here that all scyllo-inositol derivatives demonstrated reduced effectiveness in preventing Abeta(42) fibrillization compared with scyllo-inositol, suggesting that scyllo-inositol interacts with Abeta(42) via key hydrogen bonds that are formed by all hydroxyl groups. Increasing the hydrophobicity of scyllo-inositol by the addition of two methoxy groups (1,4-di-O-methyl-scyllo-inositol) produced a derivative that stabilized Abeta(42) protofibrils in vitro. Prophylactic administration of 1,4-di-O-methyl-scyllo-inositol to TgCRND8 mice attenuated spatial memory impairments and significantly decreased cerebral amyloid pathology. These results suggest that Abeta aggregation can be targeted at multiple points along the kinetic pathway for the improvement of Alzheimer's disease-like pathology.

Synthesis of OSW-1 analogs with modified side chains and their antitumor activities.

Four analogs of OSW-1 (1-4) with modified side chains on the steroidal skeleton were synthesized following modification of our previous route for the total synthesis of OSW-1. Testing of the analogs against growth of tumor cells demonstrated that the 22-one function and the full length of the side chain of OSW-1 were not required for the antitumor action of OSW-1.