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Find video protocols related to scientific articles indexed in Pubmed.
Glycoconjugate vaccine containing Escherichia coli O157:H7 O-antigen linked with maltose-binding protein elicits humoral and cellular responses.
PLoS ONE
PUBLISHED: 08-19-2014
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Glycoconjugate is one of the most efficacious and safest vaccines against bacterial pathogens. Previous studies of glycoconjugates against pathogen E. coli O157:H7 focused more on the humoral responses they elicited. However, little was known about their cellular responses. In this study, we exploited a novel approach based on bacterial protein N-linked glycosylation system to produce glycoconjugate containing Escherichia coli O157:H7 O-antigen linked with maltose-binding protein and examined its humoral and cellular responses in BALB/c mice. The transfer of E. coli O157:H7 O-antigen to MBP was confirmed by western blot and MALDI-TOF MS. Mice injected with glycoconjugate O-Ag-MBP elicited serum bactericidal antibodies including anti-E. coli O157:H7 O-antigen IgG and IgM. Interestingly, O-Ag-MBP also stimulated the secretion of anti-E. coli O157:H7 O-antigen IgA in intestine. In addition, O-Ag-MBP stimulated cellular responses by recruiting Th1-biased CD4+ T cells, CD8+ T cells. Meanwhile, O-Ag-MBP induced the upregulation of Th1-related IFN-? and downregulation of Th2-related IL-4, and the upregulation of IFN-? was stimulated by MBP in a dose-dependent manner. MBP showed TLR4 agonist-like properties to activate Th1 cells as carrier protein of O-Ag-MBP. Thus, glycoconjugate vaccine E. coli O157:H7-specific O-Ag-MBP produced by bacterial protein N-linked glycosylation system was able to elicit both humoral and Th1-biased cellular responses.
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Facile synthesis of 5-hydroxy-L-lysine from D-galactose as a chiral-precursor.
Org. Biomol. Chem.
PUBLISHED: 08-13-2014
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A concise synthesis of (2S,5R) and (2S,5S)-5-hydroxy-lysine was achieved by utilizing D-galactose as a chiral-precursor with stereo retention. This synthetic strategy showcased the potential of utilizing carbohydrates as starting materials to prepare amino acids. Using the diazido intermediate, the derived ?-D-galactopyranosyl and ?-D-glucopyranosyl-(1?2)-?-D-galactosyl moieties were synthesized.
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A Precise Approach in Large Scale Core-Fucosylated Glycoproteins Identification with Low- and High-Normalized Collision Energy.
J Proteomics
PUBLISHED: 04-29-2014
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The core fucosylation (CF) of N-glycoproteins plays important roles in regulating protein functions during biological development, and it has also been shown to be up-regulated in several high metastasis cancer cell lines. Therefore, global profiling and quantitative characterization of CF-glycoproteins may reveal potent biomarkers for clinical applications. However, due to the complex fragmentation pattern of CF-glycopeptides, accurately identifying CF-glycosylation sites via mass spectrometry with high throughput remains a formidable challenge. In this study, we established a precise CF-glycosylation site identification strategy with UHPLC LTQ-Orbitrap Elite under low- and high-normalized collision energy (LHNCE) conditions. To demonstrate the feasibility of LHNCE, the CF-glycopeptides of target proteins in clinical plasma samples were applied and compared as a preliminary demonstration and resulted in the assignment of 357 unique CF-glycosylation sites from 209 CF-glycoproteins. In this study, the largest human plasma CF- glycosylation site database was constructed, and at least three-fold more CF-sites were identified compared to previously published studies. The results further demonstrated that LHNCE provides an important approach for CF-glycoprotein function studies and biomarker screening in cancer research.
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A peptide panel investigation reveals the acceptor specificity of O-GlcNAc transferase.
FASEB J.
PUBLISHED: 04-23-2014
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O-linked ?-N-acetylglucosaminylation (O-GlcNAcylation) is widely distributed on nucleocytoplasmic proteins and participates in various physiological processes. But O-GlcNAc status on numerous proteins remains unknown. To better understand this modification, computational analysis combined with experimental study was performed in this work. Structural analysis of many O-GlcNAcylation sites indicated that the modification occurred predominantly in a random coil region. Frequency analysis on many O-GlcNAcylated peptides revealed a signature sequence, PPVS/TSATT, around the modification site (underlined, position 0). Based on the sequence, a peptide panel was designed to investigate key positions affecting O-GlcNAcylation of peptides and their amino acid preference. It was indicated that 3 positions (-2, -1, and +2) had an important role for this modification, where the presence of uncharged amino acids with small side chains could confer high reactivity. The amino acid preference at key positions was further investigated on bovine crystalline ? via site-directed mutagenesis. The preferred amino acids were Pro > Ala > Gly at position -2, Ala > Thr > Val > Lys > Pro at position -1, and Ala > Gly > Arg > Glu at position +2. Altogether, these findings suggested that a substrate (peptide or protein) with Pro, Ala at position -2, and/or Val, Ala, Thr, Ser at position -1, and/or Ala, Ser, Pro, Thr, Gly at position +2 would have more chances for O-GlcNAcylation. To test the rule, 2 O-GlcNAcylation sites on sOGT (S52 and T449) were predicted and confirmed by Western blot. The present work systematically investigated the sequence signature for O-GlcNAcylation. The result will contribute to predicting the O-GlcNAc status of a protein and further functional studies.
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Amphiphilic polyethylenimine (PEI) as highly efficient non-viral gene carrier.
Org. Biomol. Chem.
PUBLISHED: 02-19-2014
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Efficient and safe gene vectors are important for gene therapy. Here, a novel family of amphiphilic polyethylenimine (PEI) LD1-PEI bearing a polar group of branched PEI 25K and four dodecyl chains was developed. Agarose gel electrophoresis was used to confirm the formation of complexes. The transfection activity of the amphiphilic carrier was evaluated in different cell lines. The in vitro study showed that LD1-PEI showed a higher transfection efficiency with improved biocompatibility than PEI 25K. Serum showed almost no or only a slight effect on LD1-PEI/DNA transfection efficiency. In summary, LD1-PEI is a promising nonviral gene carrier.
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Crystallization, preliminary X-ray crystallographic and cryo-electron microscopy analysis of a bifunctional enzyme fucokinase/L-fucose-1-P-guanylyltransferase from Bacteroides fragilis.
Acta Crystallogr F Struct Biol Commun
PUBLISHED: 02-04-2014
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Fucokinase/L-fucose-1-P-guanylyltransferase (FKP) is a bifunctional enzyme which converts L-fucose to Fuc-1-P and thence to GDP-L-fucose through a salvage pathway. The molecular weights of full-length FKP (F-FKP) and C-terminally truncated FKP (C-FKP, residues 300-949) are 105.7 and 71.7?kDa, respectively. In this study, both recombinant F-FKP and C-FKP were expressed and purified. Size-exclusion chromatography experiments and analytical ultracentrifugation results showed that both F-FKP and C-FKP are trimers. Native F-FKP protein was crystallized by the vapour-diffusion method and the crystals belonged to space group P212121 and diffracted synchrotron X-rays to 3.7?Å resolution. The crystal unit-cell parameters are a = 91.36, b = 172.03, c = 358.86?Å, ? = ? = ? = 90.00°. The three-dimensional features of the F-FKP molecule were observed by cryo-EM (cryo-electron microscopy). The preliminary cryo-EM experiments showed the F-FKP molecules as two parallel disc-shaped objects stacking together. Combining all results together, it is assumed that there are six FKP molecules in one asymmetric unit, which corresponds to a calculated Matthews coefficient of 2.19?Å(3)?Da(-1) with 43.83% solvent content. These preliminary crystallographic and cryo-EM microscopy analyses provide basic structural information on FKP.
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Preparation, antiangiogenic and antitumoral activities of the chemically sulfated glucan from Phellinus ribis.
Carbohydr Polym
PUBLISHED: 01-25-2014
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Two sulfated derivatives (PRP-S1 and PRP-S2) of a ?-glucan from Phellinus ribis with different degrees of substitution were obtained by chlorosulfonic acid method. The derivatives could block formation of new vessels in zebrafish and inhibit the proliferation of human umbilical vein endothelial cells (HUVECs). The two sulfated derivatives had remarkably high antitumor activities in vivo (in BALB/c mice inoculated with H22 hepatocellular carcinoma) as well as in vitro (against human ovary cancer SKOV-3 cells), without producing any overt signs of general toxicity. The results of immunohistochemistry assay indicated that the derivatives significantly reduced the average number of microvessel density (MVD) and inhibited the expression of vascular endothelial growth factor (VEGF) in tumor. Thus, these derivatives exhibit pronounced antiangiogenic and antitumoral properties. Except for cytotoxic effects on tumor cells, it is reasonable to expect that the antitumoral effects of PRP-S1 and PRP-S2 are mediated via their antiangiogenic properties.
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O-antigen polymerase adopts a distributive mechanism for lipopolysaccharide biosynthesis.
Appl. Microbiol. Biotechnol.
PUBLISHED: 01-18-2014
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Bacterial lipopolysaccharide (LPS) is an essential cell envelope component for gram-negative bacteria. As the most variable region of LPS, O antigens serve as important virulence determinants for many bacteria and represent a promising carbohydrate source for glycoconjugate vaccines. In the Wzy-dependent O-antigen biosynthetic pathway, the integral membrane protein Wzy was shown to be the sole enzyme responsible for polymerization of O-repeat unit. Its catalytic mechanism, however, remains elusive. Herein, Wzy was successfully overexpressed in Escherichia coli with an N-terminal His10-tag. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed that the Wzy protein exists in its native confirmation as a dimer. Subsequently, we chemo-enzymatically synthesized the substrates of Wzy, the lipid-PP-linked repeat units. Together with an optimized O-antigen visualization method, we monitored the production of reaction intermediates at varying times. We present here our result as the first biochemical evidence that Wzy functions in a distributive manner.
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A one-pot approach to bio-synthesize globotriose and its derivatives from simpler substrates.
Eur J Med Chem
PUBLISHED: 01-14-2014
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Globotriose is involved in numerous pathogenic processes and drug development strategies. Recent studies have demonstrated that globotriosylceramide could be used in colon cancer therapy and as a crucial indicator for susceptibility to HIV-1 infection. Therefore, the cost-effective and facile approaches for large-scale production of globotiose as potential drugs are highly required. Here, a multi-enzyme one-pot system containing a galactokinase (SpGalK, E.C.2.7.1.6), a UDP-glucose pyrophosphorylase (SpGalU, E.C.2.7.7.9), a ?-1,4-galactosyltransferase (LgtC, E.C. 2.4.1.44) and a commercial inorganic pyrophosphatase (PPase, EC 3.6.1.1) was designed to achieve globotriose on preparative scales. This method exploits a cheaper initial substrate, galactose, for donor UDP-galactose production. More importantly, the substrate specificity of SpGalK and SpGalU is highly promiscuous and various UDP-galactose derivatives obtained could be used as the donor substrates for LgtC. This pointcut of rapid preparation of globotriose derivatives is proposed for the first time. Finally, three globotriose analogs were achieved by this one-pot multi-enzyme system in our study.
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Chemoenzymatic synthesis of ADP-d-glycero-?-d-manno-heptose and study of the substrate specificity of HldE.
Bioorg. Med. Chem.
PUBLISHED: 01-14-2014
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An efficient one-pot three enzymes strategy for chemoenzymatic synthesis of ADP-d-glycero-?-d-manno-heptose (ADP-d, d-heptose) was reported using chemically synthesized d, d-heptose-7-phosphate and the ADP-d, d-heptose biosynthetic enzymes HldE and GmhB. Moreover, the result of investigating substrate specificity of the kinase action of HldE revealed that HldE had highly restricted substrate specificity towards structurally modified heptose-7-phosphate analogs.
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Total synthesis of thaxtomin a and its stereoisomers and findings of their biological activities.
Org. Lett.
PUBLISHED: 10-25-2013
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The first and facile total synthesis of thaxtomin A and its three stereoisomers has been achieved. The synthetic approach involves intramolecular nucleophilic cyclization of an amide toward a ketoamide group to produce a C-hydroxydiketopiperazine scaffold. The most amazing discovery was that each of the four stereoisomers of TA exhibits different phytotoxic, fungicidal, and antiviral activities.
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Efficient enzymatic synthesis of guanosine 5-diphosphate-sugars and derivatives.
Org. Lett.
PUBLISHED: 10-11-2013
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An N-acetylhexosamine 1-kinase from Bifidobacterium infantis (NahK_15697), a guanosine 5-diphosphate (GDP)-mannose pyrophosphorylase from Pyrococcus furiosus (PFManC), and an Escherichia coli inorganic pyrophosphatase (EcPpA) were used efficiently for a one-pot three-enzyme synthesis of GDP-mannose, GDP-glucose, their derivatives, and GDP-talose. This study represents the first facile and efficient enzymatic synthesis of GDP-sugars and derivatives starting from monosaccharides and derivatives.
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Probing the role of highly conserved residues forming the acceptor binding pocket of the promiscuous glycosyltransferase MGT in defining the specificity towards a panel of flavonoids.
Biochemistry Mosc.
PUBLISHED: 07-16-2013
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MGT, a macrolide UDP-glycosyltransferase from Streptomyces lividans, has been employed as a synthetic "tool kit" to synthesize a series of modified antibiotics owing to its broad substrate plasticity. Other than MGT, a number of UDP-glycosyltransferases with substrate promiscuity were also used to alter glycosylation patterns of secondary metabolites in an emerging method called "chemoenzymatic glycorandomization". However, the structural basis of tolerating variant acceptors for these glycosyltransferases is still not clear. In this study, the relationship between the amino acid residues forming the acceptor binding pocket and the specificity of an MGT was investigated in evolutionary and structural aspects. Interestingly, alterations of the volume and hydrophobic environment of the binding pocket by replacing Ile127 or Val151 with a bulky Phe conferred on the MGT novel activities for glycosylating flavonoids that are not accepted by the wild-type MGT.
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Polysaccharide-based biomaterials with on-demand nitric oxide releasing property regulated by enzyme catalysis.
Biomaterials
PUBLISHED: 06-25-2013
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The regulatory role of nitric oxide (NO) in cell signaling has been well recognized. Clinically, NO deficiency is known to be associated with severe vascular disorders, especially in patients with long-term diabetes. Exogenous compensation of NO is a promising therapeutic strategy, although the lack of stable NO compounds often lead to unsatisfactory clinical outcomes. In the present study, we report a stable comb-shaped polymer (CS-NO) using glycosylated NO compound as pendent chains and chitosan (CS) as backbone for controlled NO release. The on-demand release of NO is achieved by controlling the decomposition process of the CS-NO polymer, which is blocked by galactose and only occurs in the presence of glycosidase, making the NO releasing kinetic closely correlate with the glycosidase concentration. In addition, due to its high stability, the CS-NO polymers can also be processed into supportive membrane or injectable hydrogel, further demonstrating its clinical potential. Indeed, we report that the NO-releasing membrane inhibited platelet adhesion, prolonged activated partial thromboplastin time (APTT) as shown in the platelet-rich-plasma (PRP) assay. We also observe enhanced human umbilical vein endothelial cell growth yet suppressed vascular smooth muscle cell proliferation on the NO-contained membrane in vitro. Furthermore, in vivo administration of CS-NO solution significantly enhanced angiogenesis in diabetic mice with hind-limb ischemia. Protective effect of CS-NO was also observed against limb necrosis. Given the physiological importance of NO, the CS-NO polymer may be considered a promising option in therapeutic development against vascular disorders and diabetic feet.
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New insights into the pharmacological chaperone activity of c2-substituted glucoimidazoles for the treatment of Gaucher disease.
Chembiochem
PUBLISHED: 03-30-2013
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Mutations in acid ?-glucocerebrosidase (GCase) lead to the accumulation of the sphingolipid glucosylceramide, thereby resulting in Gaucher disease (GD). Active-site-specific competitive GCase inhibitors are effective pharmacological chaperones (PCs) that act as folding agents for mutant GCase folding in the endoplasmic reticulum. In this study, we prepared a series of glucoimidazole C2-substituent derivatives, and evaluated their inhibition and PC properties with GCase. A cell-based assay with patient-derived lymphoblasts (N370S or L444P mutations) demonstrated that administration of these compounds can significantly increase GCase activity. Interestingly, the 3,3-dimethyl-N-phenyl-4-amide-1-butyl-substituted moderate inhibitor 11 had the greatest effect on activity: 2.1-fold increase in N370S lymphoblasts at 2.5 ?M and 1.2-fold increase in L444P at 0.5 ?M following a three-day incubation. Computer docking studies and a protease protection assay were used to elucidate the ligand-enzyme interactions responsible for the chaperone activity of 11. Western blot and immuno-fluorescence assays verified restoration of GCase trafficking to the lysosome. Together, these results indicate that 11 is a promising PC for GD treatment and provide direct evidence of the mechanism of GCase chaperoning.
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Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP).
Bioorg. Med. Chem. Lett.
PUBLISHED: 03-27-2013
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Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP.
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A chemoenzymatic route to synthesize unnatural sugar nucleotides using a novel N-acetylglucosamine-1-phosphate pyrophosphorylase from Camphylobacter jejuni NCTC 11168.
Bioorg. Med. Chem. Lett.
PUBLISHED: 03-19-2013
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A novel N-acetylglucosamine-1-phosphate pyrophosphorylase was identified from Campylobacter jejuni NCTC 11168. An unprecedented degree of substrate promiscuity has been revealed by systematic studies on its substrate specificities towards sugar-1-P and NTP. The yields of the synthetic reaction of seven kinds of sugar nucleotides catalyzed by the enzyme were up to 60%. In addition, the yields of the other nine were around 20%. With this enzyme, three novel sugar nucleotide analogs were synthesized on a preparative scale and well characterized.
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One-pot three-enzyme synthesis of UDP-Glc, UDP-Gal, and their derivatives.
Carbohydr. Res.
PUBLISHED: 02-26-2013
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A UTP-glucose-1-phosphate uridylyltransferase (SpGalU) and a galactokinase (SpGalK) were cloned from Streptococcus pneumoniae TIGR4 and were successfully used to synthesize UDP-galactose (UDP-Gal), UDP-glucose (UDP-Glc), and their derivatives in an efficient one-pot reaction system. The reaction conditions for the one-pot multi-enzyme synthesis were optimized and nine UDP-Glc/Gal derivatives were synthesized. Using this system, six unnatural UDP-Gal derivatives, including UDP-2-deoxy-Galactose and UDP-GalN3 which were not accepted by other approach, can be synthesized efficiently in a one pot fashion. More interestingly, this is the first time it has been reported that UDP-Glc can be synthesized in a simpler one-pot three-enzyme synthesis reaction system.
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Antitumor effects of a novel histone deacetylase inhibitor NK-HDAC-1 on breast cancer.
Oncol. Rep.
PUBLISHED: 02-07-2013
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Histone deacetylases (HDACs) are overexpressed in various types of primary human cancer and have become attractive targets for cancer therapy. We designed and synthesized a series of new class of HDAC inhibitors (HDACi). Among these, S-(E)-3-(1-(1-(benzo[d]oxazol-2-yl)-2-methylpropyl)-1H-1,2,3-triazol-4-yl)-N-hydroxyacrylamide (NK-HDAC-1) showed potent antitumor activity. In the present study, we examined the antitumor effects of NK-HDAC-1 on breast cancer in vitro and in vivo. The inhibitory effects of NK-HDAC-1 on HDAC enzyme activity and cell growth were more potent compared to suberoylanilide hydroxamic acid (SAHA). NK-HDAC-1 caused G1 cell cycle arrest at concentrations below 0.2 µM and G2/M arrest at concentrations above 0.4 µM through p21 upregulation and cyclin D1 downregulation. NK-HADC-1 induced hyperacetylation of histone H3 and H4 around the promoter region of p21. NK-HDAC-1 promoted apoptosis in MDA-MB-231 breast cancer cells by activating both the intrinsic and the extrinsic pathway NK-HDAC-1 at doses of 3, 10 and 30 mg/kg reduced the tumor volume in MDA-MB-231 xenografts by 25.9, 48.8 and 63.6%, respectively. The results suggested that NK-HDAC-1 may be a promising therapeutic candidate in treating human breast cancer.
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Preparation of oligosaccharides by homogenous enzymatic synthesis and solid phase extraction.
Chem. Commun. (Camb.)
PUBLISHED: 09-09-2011
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This communication describes a method for enzymatic preparation of bioactive glycans, which integrated the high-efficiency of homogenous phase enzymatic reaction and fast separation of solid phase extraction.
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Enzymatic synthesis of D-sorbose and D-psicose with aldolase RhaD: effect of acceptor configuration on enzyme stereoselectivity.
Bioorg. Med. Chem. Lett.
PUBLISHED: 08-25-2011
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It was previously reported that DHAP-dependent aldolase RhaD selectively chooses L-glyceraldehyde from racemic glyceraldehyde to produce l-fructose exclusively. Contrastingly, we discovered that D-glyceraldehyde is also tolerated as an acceptor and the stereoselectivity of the enzyme is lost in the corresponding aldol addition. Furthermore, we applied this property to efficiently synthesize two rare sugars D-sorbose and D-psicose.
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Fine tuning by human CD1e of lipid-specific immune responses.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-15-2011
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CD1e is a member of the CD1 family that participates in lipid antigen presentation without interacting with the T-cell receptor. It binds lipids in lysosomes and facilitates processing of complex glycolipids, thus promoting editing of lipid antigens. We find that CD1e may positively or negatively affect lipid presentation by CD1b, CD1c, and CD1d. This effect is caused by the capacity of CD1e to facilitate rapid formation of CD1-lipid complexes, as shown for CD1d, and also to accelerate their turnover. Similar results were obtained with antigen-presenting cells from CD1e transgenic mice in which lipid complexes are assembled more efficiently and show faster turnover than in WT antigen-presenting cells. These effects maximize and temporally narrow CD1-restricted responses, as shown by reactivity to Sphingomonas paucimobilis-derived lipid antigens. CD1e is therefore an important modulator of both group 1 and group 2 CD1-restricted responses influencing the lipid antigen availability as well as the generation and persistence of CD1-lipid complexes.
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The total synthesis of immunostimulant ?-galactosylceramides from naturally configured ?-galactoside raffinose.
Org. Lett.
PUBLISHED: 08-04-2011
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The total synthesis of absolute anomeric confirmation ?-galactosylceramide analogues from raffinose is described. Using the naturally occurring ?-galactoside raffinose as the starting material, the easily maneuverable protocol without glycosylation reactions ensured the critical ?-linkage in the product and simplified the synthetic procedures. The immunostimulatory activities of the new ?-galactosylceramides were validated by both in vitro and in vivo NKT cell stimulation assays.
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Insights into role of the hydrogen bond networks in substrate recognition by UDP-GalNAc 4-epimerases.
Biochem. Biophys. Res. Commun.
PUBLISHED: 07-17-2011
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UDP-hexose 4-epimerases are critical in galactose metabolism and often important in lipopolysaccharide biosynthesis as well. Three groups of these enzymes have been reported based on their substrate specificity towards non-acetylated substrates (group 1), dual specificity towards N-acetylated and non-acetylated substrates (group 2) and specificity towards N-acetylated substrates (group 3). We recently reported the structure of a novel UDP-GalNAc 4-epimerase called WbgU and based on the structure proposed a model of specific substrate recognition by UDP-GalNAc 4-epimerases. In this work, we present an analysis of the proposed model of substrate recognition using site-directed mutagenesis of WbgU and crystal structure of the His305Ala mutant. This investigation reveals that the wild-type activity of WbgU is retained in most single-point mutants targeting the active site. However, a graded loss in activity is observed in double-and triple-point mutants with the quadruple-point mutant being completely inactive corroborating the proposed rationale of substrate recognition. Furthermore, crystal structure of the His305Ala mutant shows that the structure is significantly similar to the wild-type WbgU, albeit a loss in the critical hydrogen bond network seated at His305 and ensuing minor conformational changes. It is inferred that the specific and non-specific interactions throughout the active site confer it sufficient elasticity to sustain wild-type activity for several of the single-point mutations.
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Substrate promiscuity of N-acetylhexosamine 1-kinases.
Molecules
PUBLISHED: 07-01-2011
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N-Acetylhexosamine 1-kinase (NahK) catalyzes the direct addition of a phosphate from adenosine 5-triphosphate (ATP) to the anomeric position of N-acetylhexosamine and shows similar activity towards N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). Herein we report the cloning, characterization, and substrate specificity studies of two NahKs from Bifidobacterium infantis ATCC15697 and Bifidobacterium longum ATCC55813, respectively. A new capillary electrophoresis assay method has been developed for enzyme activity assays. Both enzymes have a good expression level in E. coli (180-185 mg/L culture) and can tolerate diverse modifications at C2 of GlcNAc and GalNAc. Various GlcNAc derivatives with C6, both C2 and C6, as well as both C2 and C3 modifications are tolerable substrates for the newly cloned NahKs. Quite interestingly, despite of their low activities toward glucose and galactose, the activities of both NahKs are much higher for mannose and some of its C2, C4, and C6 derivatives. These NahKs are excellent catalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.
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Wide sugar substrate specificity of galactokinase from Streptococcus pneumoniae TIGR4.
Carbohydr. Res.
PUBLISHED: 06-14-2011
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Galactokinases (GALK) have attracted significant research attention for their potential application in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 had a temperature optimum of 45°C, and a pH optimum of 8.0. The substrate specificity and kinetics studies revealed that GalKSpe4 had moderate activity toward glucose, in contrast with very low or no activity observed in other previously reported GALKs. Most interestingly, GalKSpe4 exhibited activity for GalNAc, which had never been recorded in other GALKs found by now. This is the first time to report that bacterial GALK can recognize GalNAc.
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Enhanced antitumor effects by chemical modified IGb3 analogues.
Mol. Cancer Ther.
PUBLISHED: 06-08-2011
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Certain glycolipid antigens for natural killer T (NKT) cells can direct the overall cytokine balance of the immune response. However, the molecular mechanism of Th1- or Th2-biased cytokine secretion by NKT cells is still unknown. Previously, we synthesized isoglobotrihexosylceramide (iGb3) analogues by introducing a hydroxyl group at C4 on the ceramide portion of iGb3 to produce 4-HO-iGb3 or to further deoxylation on the terminal galactose to produce 4-dh-iGb3. Both modified iGb3, especially 4-dh-iGb3, stimulated more IFN-? production by hepatic NKT cells, and thus elicited preferential Th1 responses. Here, we found that 4-dh-iGb3-loaded bone marrow-derived dendritic cells (DC) could significantly inhibit growth of subcutaneous melanoma and suppress lung metastasis in C57BL/6 mice compared with unmodified iGb3-loaded DCs. In investigating the mechanisms of this improved activity, we found that 4-dh-iGb3 stimulation increased STAT1 signaling by NKT cells, whereas the phosphorylation of Th2 type cytokine-associated transcription factor STAT6 signaling was not affected. Analysis of the structures of iGb3 and 4-dh-iGb3 revealed that 4-dh-iGb3 provides greater stability and affinity between glycolipid and CD1d or NKT TCR complex than iGb3. Thus, 4-dh-iGb3 can improve the antitumor effects of a DC-based vaccine possibly by stabilizing the CD1d/glycolipid/TCR complex and stimulating IFN-? signaling of NKT cells. Furthermore, chemical modification of iGb3 can elicit Th1-biased responses by NKT cells, and 4-dh-iGb3 combined with a DC vaccine may serve as a potent new NKT-based therapy against tumors and infectious diseases.
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Enzymatic synthesis of a 6-sialyl lactose analogue using a pH-responsive water-soluble polymer support.
Bioorg. Med. Chem. Lett.
PUBLISHED: 03-07-2011
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The Letter describes a strategy for the enzymatic synthesis of glycans based on a pH-responsive water-soluble polymer. In neutral condition, the polymer is water-soluble and convenient for in-solution enzymatic synthesis, whereas in acidic condition (pH lower than 4.0), the polymer disconnects with the product and becomes insoluble, which can be easily removed. A 6-Sialyl lactose analogue was synthesized as a model reaction using this approach.
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Combining carbochips and mass spectrometry to study the donor specificity for the Neisseria meningitidis ?1,3-N-acetylglucosaminyltransferase LgtA.
Bioorg. Med. Chem. Lett.
PUBLISHED: 03-05-2011
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A library of 11 UDP-N-acetylglucosamine analogs were rapidly screened for their activities as donors for the Neisseria meningitidis ?1,3-N-acetylglucosaminyltransferase (LgtA) by direct on-chip reaction and detection with SAMDI-TOF mass spectrometry. Six of the analogs were active in this assay and were analyzed by SAMDI to characterize the kinetics toward LgtA. The analysis revealed that substitutions on C-2, C-4, and C-6 affect the activity of the donors, with bulky groups at these positions decreasing affinity of the donors for the enzyme, and also revealed that activity is strongly affected by the stereochemistry at C-3, but not C-4, of the donor. The study is also significant because it demonstrates that SAMDI can be used to both profile glycosyltransferase activities and to provide a quantitative assessment of enzyme activity.
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Design and synthesis of O-GlcNAcase inhibitors via click chemistry and biological evaluations.
Carbohydr. Res.
PUBLISHED: 03-03-2011
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Protein O-GlcNAcylation has been shown to play an important role in a number of biological processes, including regulation of the cell cycle, DNA transcription and translation, signal transduction, and protein degradation. O-GlcNAcase (OGA) is responsible for the removal of O-linked ?-N-acetylglucosamine (O-GlcNAc) from serine or threonine residues, and thus plays a key role in O-GlcNAc metabolism. Potent OGA inhibitors are useful tools for studying the cellular processes of O-GlcNAc, and may be developed as drugs for the treatment neurodegenerative diseases. In this study, Cu(I)-catalyzed Click cycloaddition reactions between glycosyl azides and alkynes were exploited to generate inhibitory candidates of OGA. Enzymatic kinetic screening revealed that compound 7 was a potent competitive inhibitor of human O-GlcNAcase (K(i)=185.6 ?M). Molecular docking simulations of compound 7 into CpOGA (Clostridium perfringens OGA) suggested that strong ?-? stacking interaction between the compound and W490 considerably contributed to improving the inhibitory activity.
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Concise synthesis of 5-methoxy-6-hydroxy-2-methylchromone-7-O- and 5-hydroxy-2-methylchromone-7-O-rutinosides. Investigation of their cytotoxic activities against several human tumor cell lines.
J. Org. Chem.
PUBLISHED: 03-02-2011
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The synthesis of two novel 2-methylchromone-7-O-rutinosides is reported, and the in vitro biological activities of these compounds and their synthetic precursors have been investigated on the basis of their cytotoxicity against several human tumor cell lines. The synthesis features early stage assembly of the acidic labile glycosidic bond between sugar and 2-methylchromone aglycon under phase transfer catalyzed glycosidation conditions, whereas all the other standard glycosylation conditions specific to a wide array of rutinosyl donors bearing different anomeric leaving groups (e.g., SPh, OC(NH)CCl(3), Br, OH groups) failed to furnish any detectable products.
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Enzymatic synthesis and properties of uridine-5-O-(2-thiodiphospho)-N-acetylglucosamine.
Carbohydr. Res.
PUBLISHED: 02-24-2011
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This paper describes an enzymatic approach to obtain a thio-containing UDP-GlcNAc analog. We use an assay based on capture of the carbohydrate and analysis by mass spectrometry to quantitatively characterize the activity of this unnatural sugar donor in a LgtA-mediated glycosylation reaction.
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Synthesis of rare sugars with L-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8.
Bioorg. Med. Chem. Lett.
PUBLISHED: 02-24-2011
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We report herein a one-pot four-enzyme approach for the synthesis of the rare sugars d-psicose, d-sorbose, l-tagatose, and l-fructose with aldolase FucA from a thermophilic source (Thermus thermophilus HB8). Importantly, the cheap starting material DL-GP (DL-glycerol 3-phosphate), was used to significantly reduce the synthetic cost.
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Structure-activity relationships in a series of C2-substituted gluco-configured tetrahydroimidazopyridines as ?-glucosidase inhibitors.
Bioorg. Med. Chem.
PUBLISHED: 01-13-2011
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Inhibition of glycoside hydrolases has widespread application in treatment of diabetes, viral infections, lysosomal storage diseases and cancers. Gluco-configured tetrahydroimidazopyridines are the most potent ?-glucosidase inhibitors reported to date. Using transition state mimic strategy, a series of C2-substituted gluco-configured tetrahydroimidazopyridines were designed and synthesized. Compounds 3 (K(i)=0.64 nM) and 5 (K(i)=0.58 nM) showed stronger inhibitory potency against ?-glucosidase. Maestro 9.1 was used to study the structure-activity relationships by docking the compounds into the ?-glucosidase active sites.
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L-rhamnose antigen: a promising alternative to ?-gal for cancer immunotherapies.
ACS Chem. Biol.
PUBLISHED: 11-11-2010
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The targeting of autologous vaccines toward antigen presenting cells (APCs) via the in vivo complexation between anti ?-Gal (anti-Gal) antibodies and ?-Gal antigens presents a promising cancer immunotherapy with enhanced immunogenicity. This strategy takes advantage of the ubiquitous anti-Gal antibody in human serum. In contrast to the ?-Gal epitope, the recent identification of high titers of anti-l-rhamnose (anti-Rha) antibodies in humans reveals a new approach toward immunotherapy employing l-rhamnose (Rha) monosaccharides. In order to evaluate this simple antigen in preclinical applications, we have synthesized Rha-conjugated immunogens and successfully induced high titers of anti-Rha antibodies in wildtype mice. Moreover, our studies demonstrate for the first time that wildtype mice could replace ?1,3galactosyltransferase knockout (?1,3GT KO) mice in such antigen/antibody-mediated vaccine design when developing cancer immunotherapies.
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Introduction of aromatic group on 4-OH of ?-GalCer manipulated NKT cell cytokine production.
Bioorg. Med. Chem.
PUBLISHED: 09-15-2010
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The glycosphingolipid ?-GalCer has been found to influence mammalian immune system significantly through the natural killer T cells. Unfortunately, the pre-clinical and clinical studies revealed several critical disadvantages that prevented the therapeutic application of ?-GalCer in treating cancer and other diseases. Recently, the detailed illustration of the CD1d/?-GalCer/NKT TCR complex crystal structural, together with other latest structural and biological understanding on glycolipid ligands and NKT cells, provided a new platform for developing novel glycolipid ligands with optimized therapeutic effects. Here, we designed a series of novel aromatic group substituted ?-GalCer analogues. The biological activity of these analogues was characterized and the results showed the unique substitution group manipulated the immune responses of NKT cells. Computer modeling and simulation study indicated the analogues had unique binding mode when forming CD1d/glycolipid/NKT TCR complex, comparing to original ?-GalCer.
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Highly efficient chemoenzymatic synthesis of ?1-3-linked galactosides.
Chem. Commun. (Camb.)
PUBLISHED: 09-10-2010
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A novel D-galactosyl-?1-3-N-acetyl-D-hexosamine phosphorylase cloned from Bifidobacterium infantis (BiGalHexNAcP) was used with a recombinant E. coli K-12 galactokinase (GalK) for efficient one-pot two-enzyme synthesis of T-antigens, galacto-N-biose (Gal?1-3GalNAc), lacto-N-biose (Gal?1-3GlcNAc), and their derivatives.
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Characterization of WbiQ: An ?1,2-fucosyltransferase from Escherichia coli O127:K63(B8), and synthesis of H-type 3 blood group antigen.
Biochem. Biophys. Res. Commun.
PUBLISHED: 08-10-2010
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Escherichia coli O127:K63(B8) possesses high human blood group H (O) activity due to its O-antigen repeating unit structure. In this work, the wbiQ gene from E. coli O127:K63(B8) was expressed in E. coli BL21 (DE3) and purified as a fusion protein containing an N-terminal GST affinity tag. Using the GST-WbiQ fusion protein, the wbiQ gene was identified to encode an ?1,2-fucosyltransferase using a radioactivity based assay, thin-layer chromatography assay, as well confirming product formation by using mass spectrometry and NMR spectroscopy. The fused enzyme (GST-WbiQ) has an optimal pH range from 6.5 to 7.5 and does not require the presence of a divalent metal to be enzymatically active. WbiQ displays strict substrate specificity, displaying activity only towards acceptors that contain Gal-?1,3-GalNAc-?-OR linkages; indicating that both the Gal and GalNAc residues are vital for enzymatic activity. In addition, WbiQ was used to prepare the H-type 3 blood group antigen, Fuc-?1,2-Gal-?1,3-GalNAc-?-OMe, on a milligram scale.
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Chemoenzymatic synthesis of uridine 5-diphospho-2-acetonyl-2-deoxy-alpha-D-glucose as C(2)-carbon isostere of UDP-GlcNAc.
J. Org. Chem.
PUBLISHED: 04-14-2010
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2-ketoGlc, which is the C(2)-carbon isostere of GlcNAc, is a novel GlcNAc analogue with a ketone group. The corresponding glycosyltransferase donor substrate, UDP-2-ketoGlc, is necessary for synthesizing 2-ketoGlc-containing molecules and is thus highly important for metabolic polysaccharide remodeling and engineering. We report here the first chemoenzymatic synthesis of UDP-2-ketoGlc using our two-enzyme (NahK and GlmU) system in vitro.
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Efficient synthesis of galactosylceramide analogues for iNKT cell stimulation.
Bioorg. Med. Chem. Lett.
PUBLISHED: 04-12-2010
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Glycolipids are potential antigens for iNKT cells recognition and demonstrate important roles in both innate and adaptive immunity. However, the difficulties in the preparation of pure configuration defined glycolipids limit the exploration of their different profiles in activating iNKT cells. We report here a concise and stereospecific preparation of novel galactosylceramide analogues by oxime ligation. This strategy would provide an efficient way to generate varied glycolipid analogues with either synthetic or natural carbohydrates for biological evaluations.
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Overexpression and topology of bacterial oligosaccharyltransferase PglB.
Biochem. Biophys. Res. Commun.
PUBLISHED: 03-17-2010
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Campylobacter jejuni contains a post-translational N-glycosylation system in which a STT3 homologue, PglB, functions as the oligosaccharyltransferase. Herein, we established a method for obtaining relatively large quantities of homogenous PglB proteins. PglB was overexpressed in Escherichia coli C43(DE3) at a level of 1 mg/L cell cultures. The activity of purified PglB was verified using a chemically synthesized sugar donor: N-acetylgalactosamine-diphospho-undecaprenyl (GalNAc-PP-Und) and a synthesized peptide acceptor. The result confirms that PglB is solely responsible for the oligosaccharyltransferase activity and complements the finding that PglB exhibits relaxed sugar substrate specificity. In addition, we performed the topology mapping of PglB using the PhoA/LacZ fusion method. The topological model shows that PglB possesses 11 transmembrane segments and two relatively large periplasmic regions other than the C-terminal domain, which is consistent with the proposal of the common N(cyt)-C(peri) topology with 11 transmembrane segments for the STT3 family proteins.
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Enzymatic route to preparative-scale synthesis of UDP-GlcNAc/GalNAc, their analogues and GDP-fucose.
Nat Protoc
PUBLISHED: 03-11-2010
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Enzymatic synthesis using glycosyltransferases is a powerful approach to building polysaccharides with high efficiency and selectivity. Sugar nucleotides are fundamental donor molecules in enzymatic glycosylation reactions by Leloir-type glycosyltransferases. The applications of these donors are restricted by their limited availability. In this protocol, N-acetylglucosamine (GlcNAc)/N-acetylgalactosamine (GalNAc) are phosphorylated by N-acetylhexosamine 1-kinase (NahK) and subsequently pyrophosphorylated by N-acetylglucosamine uridyltransferase (GlmU) to give UDP-GlcNAc/GalNAc. Other UDP-GlcNAc/GalNAc analogues can also be prepared depending on the tolerance of these enzymes to the modified sugar substrates. Starting from L-fucose, GDP-fucose is constructed by one bifunctional enzyme L-fucose pyrophosphorylase (FKP) via two reactions.
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In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz.
Nat. Chem. Biol.
PUBLISHED: 02-03-2010
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Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria-host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach that enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared undecaprenyl-diphospho-N-acetyl-D-galactosamine, the E. coli O86 oligosaccharide repeating unit was assembled by means of sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy using a chaperone coexpression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of more substrates revealed a defined mode of recognition for Wzy toward the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well defined system for further underpinning molecular details of this biosynthetic pathway.
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Targeting wall techoic acid biosynthesis: an in vivo based high-throughput screen for small molecule inhibitors.
ACS Chem. Biol.
PUBLISHED: 11-06-2009
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Identification of uncommon targets for inhibition, such as virulence factors, represents an emerging approach for combating the problem of antibiotic resistance among bacteria. Unfortunately, the lack of effective systems for the discovery and evaluation of inhibitors for such targets has considerably slowed progress. A recent article in ACS Chemical Biology, however, details the development of an in vivo based high-throughput screening strategy for identification of small molecule inhibitors of wall techoic acid biosynthesis.
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Enzymatic synthesis of UDP-GlcNAc/UDP-GalNAc analogs using N-acetylglucosamine 1-phosphate uridyltransferase (GlmU).
Chem. Commun. (Camb.)
PUBLISHED: 10-20-2009
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Reports the generation of a library composed of UDP-GlcNAc/UDP-GalNAc and investigates the substrate specificity of Escherichia coli GlcNAc-1-P uridyltransferase GlmU.
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Perchlorotrityl radical-fluorophore conjugates as dual fluorescence and EPR probes for superoxide radical anion.
Bioorg. Med. Chem.
PUBLISHED: 09-12-2009
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Perchlorotrityl radicals, mono-substituted with a fluorophore using an amide linker of varying chain length, were synthesized and characterized. Electron paramagnetic resonance (EPR) spectroscopic study indicated free-electron coupling with the aromatic hydrogen nuclei and long-range coupling with the methylene hydrogens of the linker group. Reactivity of the fluorophore-conjugated trityls with superoxide radical anion showed quenching of EPR signal and enhancement of fluorescence emission spectrum. This work presents the first example of a perchlorotrityl-fluorophore conjugate that can potentially be employed as a dual probe for the detection of superoxide under oxidative stress-mediated conditions in biological systems.
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N-terminal deletion of peptide:N-glycanase results in enhanced deglycosylation activity.
PLoS ONE
PUBLISHED: 08-01-2009
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Peptide:N-glycanase catalyzes the detachment of N-linked glycan chains from glycopeptides or glycoproteins by hydrolyzing the beta-aspartylglucosaminyl bond. Peptide:N-glycanase in yeast binds to Rad23p through its N-terminus. In this study, the complex formed between Peptide:N-glycanase and Rad23p was found to exhibit enhanced deglycosylation activity, which suggests an important role for this enzyme in the misfolded glycoprotein degradation pathway in vivo. To investigate the role of this enzyme in this pathway, we made stepwise deletions of the N-terminal helices of peptide:N-glycanase. Enzymatic analysis of the deletion mutants showed that deletion of the N-terminal H1 helix (Png1p-DeltaH1) enhanced the deglycosylation activity of N-glycanase towards denatured glycoproteins. In addition, this mutant exhibited high deglycosylation activity towards native glycoproteins. Dynamic simulations of the wild type and N-terminal H1 deletion mutant implied that Png1p-DeltaH1 is more flexible than wild type Png1p. The efficient deglycosylation of Png1p-DeltaH1 towards native and non-native glycoproteins offers a potential biotechnological application.
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Systematic study on the broad nucleotide triphosphate specificity of the pyrophosphorylase domain of the N-acetylglucosamine-1-phosphate uridyltransferase from Escherichia coli K12.
Bioorg. Med. Chem. Lett.
PUBLISHED: 07-26-2009
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N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) from Escherichia coli K12 is a bifunctional enzyme that catalyzes both the acetyltransfer and uridyltransfer reactions in the prokaryotic UDP-GlcNAc biosynthetic pathway. In this study, we report the broad substrate specificity of the pyrophosphorylase domain of GlmU during its uridyltransfer reaction and the substrate priority is ranked in the following order: UTP > dUTP > dTTP > CTP > dATP/dm(6) ATP. This pyrophosphorylase domain of GlmU is also a tool to synthesize UDP-GlcNAc analogs, two examples of which were synthesized herein in multiple mg scale in vitro.
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Substrate specificity of N-acetylhexosamine kinase towards N-acetylgalactosamine derivatives.
Bioorg. Med. Chem. Lett.
PUBLISHED: 06-26-2009
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We report herein a bacterial N-acetylhexosamine kinase, NahK, with broad substrate specificity towards structurally modified GalNAc analogues, and the production of a GalNAc-1-phosphate library using this kinase.
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Adaptability of the semi-invariant natural killer T-cell receptor towards structurally diverse CD1d-restricted ligands.
EMBO J.
PUBLISHED: 06-17-2009
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The semi-invariant natural killer (NK) T-cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the alpha-chain of the NKTcr is invariant, the beta-chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an alpha-linked monosaccharide (alpha-galactosylceramide and alpha-galactosyldiacylglycerol) and the beta-linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their beta-chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including alpha-galactosylceramide, the structurally similar alpha-galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr beta-chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr beta-chain allows these cells to recognise unique aspects of structurally diverse CD1d-restricted ligands.
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Computational structure activity relationship studies on the CD1d/glycolipid/TCR complex using AMBER and AUTODOCK.
J Chem Inf Model
PUBLISHED: 05-13-2009
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The human CD1d protein presents a wide range of lipids to the TCR of invariant natural killer T cells (iNKT). Alpha-GalCer is one of the most potent iNKT stimulatory ligands presented by CD1d. The lipid portion of this ligand has been extensively investigated over the course of the past few years; however, the sugar portion of the ligand has received minimal attention. The following research focuses on computationally analyzing the recently crystallized CD1d/alpha-GalCer/TCR tertiary complex by molecular dynamics simulations using AMBER along with studying the structure activity relationship of the sugar headgroup also by simulation and docking using Autodock for a variety of alpha-GalCer analogs. The results show that the crystal structure is stable under simulation making it an accurate representation of the CD1d/alpha-GalCer/TCR complex and that modifications to the C2 and C3 positions of the sugar are not tolerated by the tertiary complex, whereas modifications to the C4 position are tolerated.
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A chemoenzymatic route to N-acetylglucosamine-1-phosphate analogues: substrate specificity investigations of N-acetylhexosamine 1-kinase.
Chem. Commun. (Camb.)
PUBLISHED: 04-24-2009
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Reports an efficient chemoenzymatic production of an N-acetylhexosamine 1-phophate analogues library by N-acetylhexosamine 1-kinase (NahK) and describes the respective substrate specificity on this enzyme.
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Characterization and synthetic application of a novel beta1,3-galactosyltransferase from Escherichia coli O55:H7.
Bioorg. Med. Chem.
PUBLISHED: 04-15-2009
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A beta1,3-galactosyltransferase (WbgO) was identified in Escherichia coli O55:H7. Its function was confirmed by radioactive activity assay and structure analysis of the disaccharide synthesized with the recombinant enzyme. WbgO requires a divalent metal ion, either Mn(2+) or Mg(2+), for its activity and is active between pH 6.0-8.0 with a pH optimum of 7.0. N-acetylglucosamine (GlcNAc) and oligosaccharides with GlcNAc at the non-reducing end were shown to be its preferred substrates and it can be used for the synthesis of type 1 glycan chains from these substrates. Together with a recombinant bacterial GlcNAc-transferase, benzyl beta-lacto-N-tetraoside was synthesized with the purified WbgO to demonstrate the synthetic utility of WbgO.
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Remodeling bacterial polysaccharides by metabolic pathway engineering.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 02-26-2009
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Introducing structural modifications into biomolecules represents a powerful approach to dissect their functions and roles in biological processes. Bacterial polysaccharides, despite their rich structural information and essential roles in bacterium-host interactions and bacterial virulence, have largely been unexplored for in vivo structural modifications. In this study, we demonstrate the incorporation of a panel of monosaccharide analogs into bacterial polysaccharides in a highly homogenous manner via metabolic engineering of a promiscuous sugar nucleotide biosynthetic pathway. In addition, the bioorthorgonal functional groups metabolically incorporated were exploited for cell surface labeling using in vitro selective chemical ligation reactions. In summary, our study presents a general, facile and effective approach for in vivo generation of novel tailor-made bacterial polysaccharides.
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From D-glucose to enantiomerically pure cycloctanoses. The glycosidase inhibitory capacity of medium-ring carbasugars.
J. Org. Chem.
PUBLISHED: 01-29-2009
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Exhaustive dihydroxylation of the pair of cyclooctadienols consisting of 4 and 5, which are available in enantiomerically pure form from d-glucose, resulted in the formation of two diastereomeric tetraols in each case. The difference in polarity of the 6/7 and 8/9 pairs facilitated their chromatographic separation. Ensuing acetylation and PMB deprotection allowed for the assignment of relative (and ultimately absolute) stereochemistry to the resulting monohydric alcohols on the basis of J(HH) analysis of their (1)H NMR spectra. The highly functionalized exomethylenecyclooctanes 14-17, which were derived by periodinane oxidation and Wittig olefination, were further elaborated by hydroboration and global deprotection. The eight members of the cyclooctanose family of carbasugars and their precursor intermediates consistently showed patterns of J(HH) values in line with the contiguous stereochemical relationships. Also assayed was their specific inhibitory behavior toward glycosidases.
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Analysis of Recombinant CD24 Glycans by MALDI-TOF-MS Reveals Prevalence of Sialyl-T Antigen.
Am J Biomed Sci
PUBLISHED: 01-01-2009
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CD24 is a glycosyl-phosphatidyl-inositol linked glycoprotein expressed in a broad range of cell types and is heavily glycosylated. It has been found to be over expressed in cancers and tumors and is also a costimulatory molecule. Therefore, this study was carried out to define the structures of the carbohydrates associated with the CD24 recombinant protein. The CD24 glycoproteins oligosaccharides were released by chemical and enzymatic means prior to being analyzed by MALDI-TOF-MS. The results obtained showed that CD24 is both N- and O-glycosylated. The major oligosaccharides were found to be Neu5Ac?-2,3/6Gal?-1,3GalNAc, NeuAc(2)Gal ?-1,3GalNAc(1) (O-glycans), GalNAc(2)GlcNAc(2)Man(3)Fuc(1), Gal(1)GalNAc(2)GlcNAc(2)Man(3)Fuc(1), and Gal(2)GalNAc(2)GlcNAc(2)Man(3)Fuc(1) (N-glycans). The results showed that Neu5Ac?-2,3/6Gal?-1,3GalNAc (sialyl-tumor antigen, sT), a cancer-associated carbohydrate, was the most abundant glycan associated with CD24. This result raised the intriguing possibility that CD24 may be a major carrier of the sialyl-T abundantly found in cancer cells.
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Comparing the acceptor promiscuity of a Rosa hybrida glucosyltransferase RhGT1 and an engineered microbial glucosyltransferase OleD(PSA) toward a small flavonoid library.
Carbohydr. Res.
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Glycosylation is a widespread modification of plant secondary metabolites, and catalyzed by a superfamily of enzymes called UDP-glycosyltransferases (UGTs). UGTs are often involved in late biosynthetic steps and show broad substrate specificity or regioselectivity. In this study, the acceptor promiscuity of a Rosa hybrid UGT RhGT1 and an evolved microbial UGT OleD(PSA) toward a small flavonoid library was probed and compared. Interestingly, RhGT1 showed comparable acceptor promiscuity in comparison with OleD(PSA), though the acceptor binding pocket of the latter is much more open and large. This clearly indicates that stabilization of the acceptor position by suitable hydrophobic interactions is important for the specificity or regioselectivity determination as well as overall fit of the acceptor into a big enough binding pocket. This also poses a challenge for structure-based UGT engineering to alter the glucosylation pattern of flavonoids.
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A safe and facile route to imidazole-1-sulfonyl azide as a diazotransfer reagent.
Org. Lett.
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A facile approach to the diazotransfer reagent of imidazole-1-sulfonyl azide was reported. The procedure was well optimized to clarify potential explosion risks. A high production yield as well as small batch variation was achieved even without careful pretreatment of reagents and solvents. HPLC and NMR methods to monitor the process were provided. These features made this protocol suitable for large scale preparation in academia and industry as well.
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Elevation of cellular O-GlcNAcylation level by a potent and selective O-GlcNAcase inhibitor based on tetrahydroimidazopyridine scaffold.
Bioorg. Med. Chem. Lett.
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Protein O-GlcNAc glycosylation is a ubiquitous post-translational modification in metazoans. O-GlcNAcase (OGA), which is responsible for removing O-GlcNAc from serine or threonine residues, plays a key role in O-GlcNAc metabolism. Potent and selective O-GlcNAcase (OGA) inhibitors are useful tools for investigating the role of this modification in a broad range of cellular processes, and may also serve as drug candidates for treatment of neurodegenerative diseases. Biological screening of the gluco-configured tetrahydroimidazopyridine derivatives identified a compound as a potent and competitive inhibitor of human O-GlcNAcase (OGA) with a K(i) of 5.9 ?M, and it also displayed 28-fold selectivity for human OGA over human lysosomal ?-hexosaminidase A (Hex A, K(i)=163 ?M). In addition, cell-based assay revealed that this compound was cell-permeant and effectively induced cellular hyper-O-GlcNAcylation at 10 ?M concentration.
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Development of aminoglycoside antibiotics by carbohydrate chemistry.
Mini Rev Med Chem
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With the development of glycomics, more and more carbohydrate mimetics were used to investigate the interactions between carbohydrate-proteins, especially in physiological and pathological processes, molecular recognition, signal transduction, cell communication, cell differentiation and developmental events. Recently, because of the drug-resistance of microorganisms and the development of antibiotics, the interactions between carbohydrate mimetics and RNAs are becoming hot issue. Aminoglycosides, one family of important antibiotics, can bind with 30S subunits of rRNA to prevent the normal translations of proteins, inhibit the proteins involving in the drug-resistance. In this review, the latest advances in development and applications of aminoglycosides are summarized and the detailed descriptions on the SAR study (Structure-activity relationship) of aminoglycoside derivatives are discussed.
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Discovery of glycosyltransferases using carbohydrate arrays and mass spectrometry.
Nat. Chem. Biol.
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Glycosyltransferases catalyze the reaction between an activated sugar donor and an acceptor to form a new glycosidic linkage. Glycosyltransferases are responsible for the assembly of oligosaccharides in vivo and are also important for the in vitro synthesis of these biomolecules. However, the functional identification and characterization of new glycosyltransferases is difficult and tedious. This paper describes an approach that combines arrays of reactions on an immobilized array of acceptors with an analysis by mass spectrometry to screen putative glycosyltransferases. A total of 14,280 combinations of a glycosyltransferase, an acceptor and a donor in four buffer conditions were screened, leading to the identification and characterization of four new glycosyltransferases. This work is notable because it provides a label-free method for the rapid functional annotation of putative enzymes.
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Synthesis of flavonol 3-O-glycoside by UGT78D1.
Glycoconj. J.
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Glycosylation is an important method for the structural modification of various flavonols, resulting in the glycosides with increased solubility, stability and bioavailability compared with the corresponding aglycone. From the physiological point of view, glycosylation of plant flavonoids is of importance and interest. However, it is notoriously complicated that flavonols such as quercetin, kaempferol and myricetin, are glucosylated regioselectively at the specific position by chemical method. Compared to the chemical method, enzymatic synthesis present several advantages, such as mild reaction condition, high stereo or region selectivity, no protection/deprotection and high yield. UGT78D1 is a flavonol-specific glycosyltransferase, responsible for transferring rhamnose or glucose to the 3-OH position in vitro. In this study, the activity of UGT78D1 was tested against 28 flavonoids acceptors using UDP-glucose as donor nucleoside in vitro, and 5 acceptors, quercetin, myricetin, kaempferol, fisetin and isorhamnetin, were discovered to be glucosylated at 3-OH position. Herein, the small-scale 3-O-glucosylated quercetin, kaempferol and myricetin were synthesized by UGT78D1 and their chemical structures were confirmed by (1)H and (13)C nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS).
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One-pot four-enzyme synthesis of ketoses with fructose 1,6-bisphosphate aldolases from Staphylococcus carnosus and rabbit muscle.
Carbohydr. Res.
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By the action of D-fructose 1,6-bisphosphate aldolases (FruA) from rabbit muscle and Staphylococcus carnosus, various ketoses were synthesized from glyceraldehydes or other aliphatic aldehydes as acceptors in a one-pot four-enzyme system.
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The impact of click chemistry in medicinal chemistry.
Expert Opin Drug Discov
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The copper(I)-catalyzed 1,3-dipolar cycloaddition of alkynes and azides to form 1,2,3-triazoles is the most popular reaction in click chemistry. This reaction is also near-perfect, in terms of its robustness, due to the high degree of reliability and complete specificity. Furthermore, this reaction has been used increasingly in drug discovery, because the formed 1,2,3-triazole can act as both a bioisostere and a linker.
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Investigation of the nucleotide triphosphate substrate specificity of Homo sapiens UDP-N-acetylgalactosamine pyrophosphorylase (AGX1).
Bioorg. Med. Chem. Lett.
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Nucleotide sugars are essential glycosyl donors for Leloir-type glycosyltransferases. The UDP-N-acetylgalactosamine pyrophosphorylase (UDP-GalNAc PP; AGX1) from Homo sapiens catalyzes the synthesis of UDP-N-acetylgalactosamine from N-acetylgalactosamine 1-phosphate and UTP. In this Letter, we systematically studied nucleotide substrate specificity of AGX1 during its uridyltransfer reaction, and described the capability of AGX1 to catalyze dUTP and dTTP to their corresponding nucleotide sugars for the first time. Furthermore, using such a eukaryotic enzyme, we synthesized dUDP-GalNAc and dTDP-GalNAc in multiple mg scale in vitro efficiently and rapidly.
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Synthesis and biological activities of a 3-azido analogue of Doxorubicin against drug-resistant cancer cells.
Int J Mol Sci
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Doxorubicin (DOX), an anthracycline antibiotic, is one of the most active anticancer chemotherapeutic agents. The clinical use of DOX, however, is limited by the dose-dependant P-glycoprotein (P-gp)-mediated resistance. Herein, a 3-azido analogue of DOX (ADOX) was prepared from daunorubicin (DNR). ADOX exhibited potent antitumor activities in drug-sensitive (MCF-7 and K562) and drug-resistant cell lines (MCF-7/DNR, K562/DOX), respectively. The drug resistance index (DRI) values of ADOX were much lower than that of DOX. The cytotoxicity experiments of ADOX or DOX against K562/DOX, with or without P-gp inhibitor, indicated that ADOX circumvents resistance by abolishing the P-gp recognition. This conclusion was further supported by drug influx/efflux flow cytometry experiments, as well as by molecular docking of ADOX to P-gp. In vivo animal tests, ADOX exhibited higher activity and less toxicity than DOX. The current data warranted ADOX for additional pre-clinical evaluations for new drug development.
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Discovery and extensive in vitro evaluations of NK-HDAC-1: a chiral histone deacetylase inhibitor as a promising lead.
J. Med. Chem.
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Herein, further SAR studies of lead compound NSC746457 (Shen, J.; Woodward, R.; Kedenburg, J. P.; Liu, X. W.; Chen, M.; Fang, L. Y.; Sun; D. X.; Wang. P. G. J. Med. Chem. 2008, 51, 7417-7427) were performed, including the replacement of the trans-styryl moiety with a 2-substituted benzo-hetero aromatic ring and the introduction of a substituent onto the central methylene carbon. A promising chiral lead, S-(E)-3-(1-(1-(benzo[d]oxazol-2-yl)-2-methylpropyl)-1H-1,2,3-triazol-4-yl)-N-hydroxyacrylamide (12, NK-HDAC-1), was discovered and showed about 1 order of magnitude more potency than SAHA in both enzymatic and cellular assays. For the in vitro safety tests, NK-HDAC-1 was far less toxic to nontransformed cells than tumor cells and showed no significant inhibition activity against CYP-3A4. The pharmaceutical properties (LogD, solubility, liver micrsomal stability (t1/2), plasma stability (t1/2), and apparent permeability) strongly suggested that NK-HDAC-1 might be superior to SAHA in bioavailability and in vivo half-life.
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Discovery and structural insight of a highly selective protein kinase inhibitor hit through click chemistry.
Chem. Commun. (Camb.)
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Novel bisaryl maleimide derivatives to mimic natural kinase inhibitors were prepared through click chemistry. A highly selective hit was discovered in a 124-kinase-assay, and docking studies revealed a ?-? stacking interaction with the Phe67 at the P-loop of GSK-3? kinase.
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Efficient siRNA delivery using a polyamidoamine dendrimer with a modified pentaerythritol core.
Pharm. Res.
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Delivery of siRNA into cells remains a critical challenge. Our lab has shown a novel polyamidoamine (PAMAM) dendrimer with modified pentaerythritol derivative core (PD dendrimer) to exhibit high plasmid DNA transfection efficiency and low cytotoxicity. Here, we evaluate PD dendrimer as a siRNA carrier.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.