RESUMEN
Multidrug resistance in Pseudomonas aeruginosa is a noticeable and ongoing major obstacle for inhibitor design. In P. aeruginosa, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) acetyltransferase (PaLpxA) is an essential enzyme of lipid A biosynthesis and an attractive drug target. PaLpxA is a homotrimer, and the binding pocket for its substrate, UDP-GlcNAc, is positioned between the monomer A-monomer B interface. The uracil moiety binds at one monomer A, the GlcNAc moiety binds at another monomer B, and a diphosphate form bonds with both monomers. The catalytic residues are conserved and display a similar catalytic mechanism across orthologs, but some distinctions exist between pocket sizes, residue differences, substrate positioning and specificity. The analysis of diversified pockets, volumes, and ligand positions was determined between orthologues that could aid in selective inhibitor development. Thenceforth, a complex-based pharmacophore model was generated and subjected to virtual screening to identify compounds with similar pharmacophoric properties. Docking and general Born-volume integral (GBVI) studies demonstrated 10 best lead compounds with selective inhibition properties with essential residues in the pocket. For biological access, these scaffolds complied with the Lipinski rule, no toxicity and drug likeness properties, and were considered as lead compounds. Hence, these scaffolds could be helpful for the development of potential selective PaLpxA inhibitors.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Aciltransferasas/metabolismo , Secuencia de Aminoácidos/genética , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/metabolismo , Especificidad por Sustrato/genética , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
A highly efficient di-C-glycosyltransferase GgCGT was discovered from the medicinal plant Glycyrrhiza glabra. GgCGT catalyzes a two-step di-C-glycosylation of flopropione-containing substrates with conversion rates of >98%. To elucidate the catalytic mechanisms of GgCGT, we solved its crystal structures in complex with UDP-Glc, UDP-Gal, UDP/phloretin, and UDP/nothofagin, respectively. Structural analysis revealed that the sugar donor selectivity was controlled by the hydrogen-bond interactions of sugar hydroxyl groups with D390 and other key residues. The di-C-glycosylation capability of GgCGT was attributed to a spacious substrate-binding tunnel, and the G389K mutation could switch di- to mono-C-glycosylation. GgCGT is the first di-C-glycosyltransferase with a crystal structure, and the first C-glycosyltransferase with a complex structure containing a sugar acceptor. This work could benefit the development of efficient biocatalysts to synthesize C-glycosides with medicinal potential.
Asunto(s)
Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Glycyrrhiza/enzimología , Clonación Molecular , Cristalografía por Rayos X , Glicosilación , Glicosiltransferasas/genética , Glycyrrhiza/genética , Ligandos , Modelos Moleculares , Floretina/química , Floretina/metabolismo , Especificidad por Sustrato , Transcriptoma , Uridina Difosfato Galactosa/química , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato Ácido Glucurónico/química , Uridina Difosfato Ácido Glucurónico/metabolismo , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismo , Uridina Difosfato Xilosa/química , Uridina Difosfato Xilosa/metabolismoRESUMEN
LpxA, the first enzyme in the biosynthetic pathway for the Lipid A component of the outer membrane lipopolysaccharide in Gram-negative bacteria, is a potential target for novel antibacterial drug discovery. A fluorescence polarization assay was developed to facilitate high-throughput screening for competitive inhibitors of LpxA. The assay detects displacement of a fluorescently labeled peptide inhibitor, based on the previously reported inhibitor peptide 920, by active site ligands. The affinity of the fluorescent ligand was increased ~10-fold by acyl carrier protein (ACP). Competition with peptide binding was observed with UDP-N-acetylglucosamine (IC(50) ~6 mM), UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine (IC(50) ~200 nM), and DL-3-hydroxymyristic acid (IC(50) ~50 µM) and peptide 920 (IC(50) ~600 nM). The IC(50)s were not significantly affected by the presence of ACP.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Polarización de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Proteína Transportadora de Acilo/metabolismo , Aciltransferasas/química , Unión Competitiva , Dominio Catalítico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Concentración 50 Inhibidora , Ligandos , Lípido A/metabolismo , Ácidos Mirísticos/química , Ácidos Mirísticos/metabolismo , Péptidos/química , Péptidos/metabolismo , Uridina Difosfato N-Acetilglucosamina/análogos & derivados , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
Whether the hexosamine biosynthesis pathway acts as a nutrient-sensing pathway is still unclear. Glucose is directed into this pathway by GFAT. Because the activity of GFAT is tightly regulated, we examined whether UDP-hexosamine levels can increase significantly and dose-dependently in response to elevated glucose concentrations. In glucosamine-treated 3T3-L1 adipocytes, inhibition of insulin-stimulated glucose uptake was highly correlated with UDP-hexosamine levels (r = -0.992; p < 0.0001 for UDP-GlcNAc and r = -0.996; p < 0.0001 for UDP-GalNAc). Incubation of 3T3-L1 adipocytes with 0.1 microM insulin for 24 h in medium containing 1 and 5 mM glucose increased the rate of glucose uptake by 365% and 175% compared to untreated cells, respectively. This increase was not observed when the cells were incubated for 24 h with insulin in medium containing 10 or 25 mM glucose. However, treatment of cells with insulin and 1, 5, 10, or 25 mM glucose resulted in similar increases in levels of UDP-GlcNAc and UDP-GalNAc that always amounted to approx 30-40% above baseline values. This led us to conclude that despite exposure of adipocytes to conditions of extreme and prolonged glucose disposal, the increases in cellular UDP-hexosamines were minimal and not dependent on the extracellular glucose concentration. Taken together, our results are in line with the hypothesis that in glucosamine-treated adipocytes UDP-hexosamines influence insulin-stimulated glucose uptake. However, our observations in glucose-treated adipocytes argue against the possibility that UDP-hexosamines function as a nutrient-sensor, and question the role of the hexosamine biosynthesis pathway in the pathogenesis of insulin resistance.
Asunto(s)
Adipocitos/metabolismo , Hexosaminas/metabolismo , Células 3T3-L1 , Animales , Desoxiglucosa/metabolismo , Glucosamina/farmacología , Glucosa/metabolismo , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Ratones , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
Protein glycosylation is a common and important process that can alter the stability, half-life, biological activity and receptor recognition of target molecules. We have identified a new putative mouse UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family member, termed GalNAc-T10/ppGaNTase-T10 (gene symbol Galnt10), and determined its expression pattern in mouse CNS using in situ hybridization analysis. Results demonstrated predominant expression of Galnt10 in several distinct hypothalamic, thalamic and amygdaloid nuclei. The most abundant hybridization levels were observed in the paraventricular, ventromedial and arcuate nuclei of the hypothalamus, the anterodorsal and parafascicular nuclei of the thalamus and the central, basomedial and medial nuclei of the amygdala. Expression of Galnt10 was also detected in cerebral cortex, lateral septum, habenula and hippocampus. The localization of this putative glycosyltransferase in distinct regions within the CNS indicates the specificity for complex protein modifications and suggests that region-specific glycosylation represents an essential process in basic biological functions.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Hipotálamo/metabolismo , N-Acetilgalactosaminiltransferasas/biosíntesis , N-Acetilgalactosaminiltransferasas/química , N-Acetilgalactosaminiltransferasas/metabolismo , Prosencéfalo/metabolismo , Tálamo/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismo , Animales , Encéfalo/metabolismo , Sistema Nervioso Central/metabolismo , Glicosilación , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Péptidos/química , ARN Mensajero/metabolismo , Distribución Tisular , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Glycosyltransferases mediate changes in glycosylation patterns which, in turn, may affect the function of glycoproteins and/or glycolipids and, further downstream, processes of development, differentiation, transformation and cell-cell recognition. Such enzymes, therefore, represent valid targets for drug discovery. We have developed a solid-phase glycosyltransferase assay for use in a robotic high-throughput format. Carbohydrate acceptors coupled covalently to polyacrylamide are coated onto 96-well plastic plates. The glycosyltransferase reaction is performed with recombinant enzymes and radiolabeled sugar-nucleotide donor at 37 degrees C, followed by washing, addition of scintillation counting fluid, and measurement of radioactivity using a 96-well beta-counter. Glycopolymer construction and coating of the plastic plates, enzyme and substrate concentrations, and linearity with time were optimized using recombinant Core 2 beta1-6-N-acetylglucosaminyltransferase (Core 2 GlcNAc-T). This enzyme catalyzes a rate-limiting reaction for expression of polylactosamine and the selectin ligand sialyl-Lewis(x) in O-glycans. A glycopolymer acceptor for beta1-6-N-acetylglucosaminyltransferase V was also designed and shown to be effective in the solid-phase assay. In a high-throughput screen of a microbial extract library, the coefficient of variance for positive controls was 9.4%, and high concordance for hit validation was observed between the Core 2 GlcNAc-T solid-phase assay and a standard solution-phase assay. The solid-phase assay format, which can be adapted for a variety of glycosyltransferase enzymes, allowed a 5-6 fold increase in throughput compared to the corresponding solution-phase assay.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/análisis , N-Acetilglucosaminiltransferasas/metabolismo , Resinas Acrílicas/metabolismo , Secuencia de Carbohidratos , Disacáridos/química , Disacáridos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Datos de Secuencia Molecular , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , N-Acetilglucosaminiltransferasas/genética , Plásticos , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Robótica , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
31P-NMR extract spectra of N-417 Small Cell Lung Cancer (SCLC) cells cultured with fluorouridine (FUrd) reveal new peaks with chemical shifts in the diphosphodiester and nucleoside triphosphate regions. These peaks were identified as FUTP, FUDP, FUDP-glucose, FUDP-glucuronate, FUDP-GlcNAc, and FUDP-GalNAc via enzymatic conversion and 19F- and 31P-NMR analysis. Distinct 19F chemical shifts were assigned for FUTP, FUDP, and the FUDP-sugars.
Asunto(s)
Carcinoma de Células Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Espectroscopía de Resonancia Magnética , Uridina/metabolismo , Flúor , Humanos , Fósforo , Células Tumorales Cultivadas , Uridina Difosfato/metabolismo , Uridina Difosfato Glucosa/metabolismo , Uridina Difosfato Ácido Glucurónico/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismo , Azúcares de Uridina Difosfato/metabolismo , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/metabolismoRESUMEN
Deficient expression of glycoinositol phospholipid (GPI) anchored proteins in affected paroxysmal nocturnal hemoglobinuria (PNH) cells has been traced to a defect in GPI anchor assembly. In a previous study (Schubert, J., Schmidt, R. E., and Medof, M. E. (1993) J. Biol. Chem., in press) we characterized the biosynthesis of putative Man-containing GPI anchor precursors in normal peripheral blood lymphocytes and investigated assembly of these intracellular GPI intermediates in CD48- affected and CD48+ unaffected T and natural killer cell lines of PNH patients. We found that affected T cells from five patients exhibited a uniform defect in which dolichol-phosphoryl-Man was synthesized but no GPI mannolipids were expressed. In this study, membranes of patients' affected T cells were labeled with UDP-[3H]GlcNAc to evaluate earlier steps in GPI synthesis, and intact cells were fused to Thy-1- murine lymphoma mutants harboring different defects in early GPI assembly to test for the presence of corresponding or complementary lesions. In all cases, affected cell membranes failed to assemble GlcNAc-inositol phospholipid, the initial precursor of GPI anchor structures, and the intact cells failed to complement class A mutants while complementing other classes. Affected polymorphonuclear leukocytes from three additional patients of different origin were then labeled with [3H]Man and the labeling patterns found to correspond to those obtained with the T lymphocytes. Taken together the data indicate that the genetic lesion in PNH cells resides in a DNA element which: 1) encodes a product required for the synthesis of GlcNAc-inositol phospholipid, 2) corresponds to that altered in class A Thy-1- murine lymphoma mutants, and 3) is commonly affected in different patients.
Asunto(s)
Acetilglucosamina/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Hemoglobinuria Paroxística/inmunología , Hemoglobinuria Paroxística/metabolismo , Linfoma/inmunología , Linfoma/metabolismo , Animales , Antígenos CD/genética , Antígeno CD48 , Membrana Celular/metabolismo , Monofosfato de Dolicol Manosa/metabolismo , Hemoglobinuria Paroxística/genética , Humanos , Células Asesinas Naturales/inmunología , Linfoma/genética , Manosa/sangre , Ratones , Mutación , Neutrófilos/inmunología , Neutrófilos/metabolismo , Trypanosoma brucei brucei/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
Confluent human retinal pigmented epithelial cells were cultured on microcarrier beads in the presence of 5.6 or 26 mmol/l glucose with or without the aldose-reductase inhibitor Sorbinil (200 microM) for 2 wk. At the end of the incubation period, perchloric acid extracts were prepared and analyzed by 31P nuclear magnetic resonance spectroscopy. As assessed by this method, the phosphorylated metabolites of cells incubated with 5.6 or 26 mmol/l glucose differed significantly in the concentrations of a number of uridine diphosphate (UDP)-conjugated monosaccharides, which were elevated two- to threefold in cells incubated in 26 mmol/l glucose over control samples. The affected metabolites were identified (through a series of spiking experiments) to be UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, and UDP-glucuronic acid. Coincubation of the cells with Sorbinil 200 microM in the presence of 26 mmol/l glucose had no effect on this accumulation. Under normal circumstances, these molecules selectively and sequentially are incorporated into the polysaccharide chains of glycosaminoglycans (GAGs), whose presence and distribution in the basement membranes is affected adversely by diabetes mellitus. These data suggest that the availability of the monosaccharide precursor is not the rate-limiting step for GAG synthesis in the presence of pathologic glucose concentrations. Thus, the lost GAG content in the basement membranes of diabetic patients may be caused by changes elsewhere in the biosynthesis and/or catabolism of the polysaccharide-linked protein molecules.
Asunto(s)
Glucosa/farmacología , Glicosaminoglicanos/metabolismo , Imidazolidinas , Epitelio Pigmentado Ocular/metabolismo , Aldehído Reductasa/antagonistas & inhibidores , Células Cultivadas , Medios de Cultivo , Humanos , Imidazoles/farmacología , Espectroscopía de Resonancia Magnética , Fósforo , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/efectos de los fármacos , Precursores de Proteínas , Uridina Difosfato Ácido Glucurónico/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
A series of experiments were conducted to identify and quantify the phosphorus metabolites of DU 145 xenografts (a human prostatic adenocarcinoma cell line grown in nude mice) using 31P MRS and HPLC. The 31P spectral characteristics of DU 145 xenografts were compared to perfused DU 145 cells and to in situ human prostatic adenocarcinomas. These studies demonstrated that both DU 145 xenografts and perfused DU 145 cells exhibited reduced levels of phosphocreatine relative to spectra of in situ human prostatic adenocarcinomas. Elevated levels of phosphomonesters (PMEs) were observed in 31P spectra of both DU 145 xenografts and in situ human prostatic adenocarcinomas. The major components of the PME resonance of DU 145 xenografts were identified as phosphocholine and phosphoethanolamine. High levels of diphosphodiesters (DPDEs) were consistently observed for both DU 145 xenografts and perfused DU 145 cells, but were absent in 31P spectra in in situ primary human adenocarcinomas. In agreement with spectroscopic results, high pressure liquid chromatographic analyses of human tissue removed at surgery contained insignificant amounts of DPDEs while DU 145 xenografts had high levels of DPDEs consisting mainly of uridine-5'-diphospho-N-acetylgalactosamine (22.4 nmol/mg protein) and uridine-5'-diphospho-N-acetylglucosamine (7.4 nmol/mg protein).
Asunto(s)
Adenocarcinoma/metabolismo , Espectroscopía de Resonancia Magnética , Fósforo/metabolismo , Neoplasias de la Próstata/metabolismo , Adenocarcinoma/patología , Anciano , Animales , Cromatografía Líquida de Alta Presión , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfocreatina/metabolismo , Neoplasias de la Próstata/patología , Células Tumorales Cultivadas , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
In liver homogenate the biosynthesis of N-acetylneuraminic acid using N-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn2+, Mn2+, La3+, Co2+, Cu2+, Hg2+, VO3-, Pb2+, Ce3+, Cd2+, Fe2+, Fe3+, Al3+, Sn2+, Cs+ and Li+) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possibly several steps of the biosynthesis of N-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmol l-1): N-acetylglucosamine kinase (inhibited by Zn2+ and vandate), UDP-N-acetylglucosamine-2'-epimerase (inhibited by Zn2+, Co2+, Cu2+, Hg2+, VO3-, Pb2+, Cd2+, Fe3+, Cs+, Li+, selenium(IV) oxide and selenite), and N-acetylmannosamine kinase (inhibited by Zn2+, Cu2+, Cd2+ and Co2+). Dose dependent measurements have shown that Zn2+, Cu2+ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2'-epimerase than vanadate. As for the N-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn2+, Cd2+, Co2+ and Cu2+. In contrast, La3+, Al3+ and Mn2+ (1 mmol l-1) did not interfere with the biosynthesis of N-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.