RESUMEN
Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Glucosamina/farmacología , Glucolípidos/farmacología , Receptor Toll-Like 4/antagonistas & inhibidores , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Adyuvantes Inmunológicos/síntesis química , Adyuvantes Inmunológicos/metabolismo , Adyuvantes Inmunológicos/toxicidad , Animales , Femenino , Glucosamina/síntesis química , Glucosamina/metabolismo , Glucosamina/toxicidad , Glucolípidos/síntesis química , Glucolípidos/metabolismo , Glucolípidos/toxicidad , Humanos , Inflamasomas/metabolismo , Interleucina-1/metabolismo , Macrófagos/efectos de los fármacos , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/metabolismoRESUMEN
We report the application of a covalent probe based on a d-glucosamine scaffold for the profiling of the bacterial pathogen Klebsiella pneumoniae. Incubation of K. pneumoniae lysates with the probe followed by electrophoretic separation and in-gel fluorescence detection allowed the generation of strain-specific signatures and the differentiation of a carbapenem-resistant strain. The labelling profile of the probe was independent of its anomeric configuration and included several low-abundance proteins not readily detectable by conventional protein staining. Initial target identification experiments by mass spectrometry suggest that target proteins include several carbohydrate-recognising proteins, which indicates that the sugar scaffold may have a role for target recognition.
Asunto(s)
Proteínas Bacterianas/genética , Colorantes Fluorescentes/química , Glucosamina/química , Klebsiella pneumoniae/genética , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes/síntesis química , Perfilación de la Expresión Génica , Glucosamina/síntesis química , Klebsiella pneumoniae/aislamiento & purificación , Estructura Molecular , Relación Estructura-ActividadRESUMEN
A series of novel N-alkyl-1-deoxynojirimycin derivatives 25 â¼ 44 were synthesised and evaluated for their in vitro α-glucosidase inhibitory activity to develop α-glucosidase inhibitors with high activity. All twenty compounds exhibited α-glucosidase inhibitory activity with IC50 values ranging from 30.0 ± 0.6 µM to 2000 µM as compared to standard acarbose (IC50 = 822.0 ± 1.5 µM). The most active compound 43 was â¼27-fold more active than acarbose. Kinetic study revealed that compounds 43, 40, and 34 were all competitive inhibitors on α-glucosidase with Ki of 10 µM, 52 µM, and 150 µM, respectively. Molecular docking demonstrated that the high active inhibitors interacted with α-glucosidase by four types of interactions, including hydrogen bonds, π-π stacking interactions, hydrophobic interactions, and electrostatic interaction. Among all the interactions, the π-π stacking interaction and hydrogen bond played a significant role in a various range of activities of the compounds.
Asunto(s)
Glucosamina/análogos & derivados , Inhibidores de Glicósido Hidrolasas/síntesis química , alfa-Glucosidasas/metabolismo , 1-Desoxinojirimicina/síntesis química , 1-Desoxinojirimicina/farmacocinética , Acarbosa/farmacología , Acarbosa/normas , Compuestos de Bencilideno/química , Glucosamina/síntesis química , Glucosamina/farmacocinética , Inhibidores de Glicósido Hidrolasas/farmacocinética , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Simulación del Acoplamiento Molecular , Relación Estructura-ActividadAsunto(s)
Glucosamina/síntesis química , Glucosamina/farmacología , Sulfatos/síntesis química , Sulfatos/farmacología , Animales , Conformación de Carbohidratos , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Glucosamina/análogos & derivados , Glucosamina/química , Ratones , Células 3T3 NIH , Sulfatos/química , Células VeroRESUMEN
The clinical effect of glucosamine, the most widely used supplement in patients with osteoarthritis, on joint pain and function improvement, is reported to be inconsistent. Inter-patient variability in the pharmacokinetics of glucosamine, especially its oral absorption, could contribute to the inconsistent clinical outcomes. To test this hypothesis, a novel but simple Hydrophilic Interaction Liquid Chromatography coupled with Charged Aerosol Detector method was developed and validated. The sample was prepared by simple protein precipitation and analysed using an amino column and acetonitrile:100â¯mM ammonium formate with gradient elution. The developed method was linear (12.5-800â¯ng/mL, r2â¯=â¯0.999) and the relative standard deviations for intra- and inter-day accuracy, precision and repeatability were all less than 6%. The sensitivity of the method (lower limit of quantitation; 12.5â¯ng/mL) allowed the quantification of endogenous and exogenous glucosamine levels in 12 patients with osteoarthritis, taking 1500â¯mg glucosamine daily. The analysis showed 120-fold variation (81.7% variance) in exogenous glucosamine levels among the patients, indicating that substantial variability in the extent of absorption and/or rate of elimination could be a possible cause for the reported inconsistent clinical outcomes. The newly-developed method was sensitive and can be used to study the pharmacokinetics of glucosamine.
Asunto(s)
Aerosoles/química , Cromatografía Líquida de Alta Presión/métodos , Glucosamina/sangre , Glucosamina/síntesis química , Plasma/química , Acetonitrilos/sangre , Acetonitrilos/química , Suplementos Dietéticos/análisis , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Colorectal cancer is a major cause of cancer-related deaths worldwide. Histologic diagnosis using biopsy samples of colorectal neoplasms is the most important step in determining the treatment methods, but these methods have limitations in accuracy and effectiveness. Herein, we report a dual-recognition two-photon probe and its application in the discrimination between human colorectal neoplasms. The probe is composed of two monosaccharides, d-glucosamine and ß-d-galactopyranoside, in a fluorophore for the monitoring of both glucose uptake and ß-gal hydrolysis. In vitro/cell imaging studies revealed the excellent selectivity and sensitivity of the probe for glucose transporter-mediated glucose uptake and ß-gal activity. Cancer-specific uptake was monitored by increased fluorescence intensity, and additional screening of cancer cells was achieved by changes in emission ratio owing to the higher activity of ß-gal. Using human colon tissues and two-photon microscopy, we found that the plot of intensity versus ratio can accurately discriminate between colorectal neoplasms in the order of cancer progression (normal, adenoma, and carcinoma).
Asunto(s)
Neoplasias Colorrectales/diagnóstico por imagen , Colorantes Fluorescentes/química , Galactósidos/química , Glucosamina/análogos & derivados , Adenoma/diagnóstico por imagen , Carcinoma/diagnóstico por imagen , Línea Celular Tumoral , Neoplasias Colorrectales/clasificación , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Colorantes Fluorescentes/efectos de la radiación , Galactósidos/síntesis química , Galactósidos/metabolismo , Galactósidos/efectos de la radiación , Glucosamina/síntesis química , Glucosamina/metabolismo , Glucosamina/efectos de la radiación , Humanos , Microscopía Fluorescente/métodos , Fotones , beta-Galactosidasa/metabolismoRESUMEN
Covalent conjugates of multiple nutrients often exhibit greater biological activities than each individual nutrient and more predictable safety profiles than completely unnatural chemical entities. Here, we report the construction and application of a focused chemical library of 308 covalent conjugates of a variety of small-molecule nutrients. Screening of the library with a reporter gene of sterol regulatory element-binding protein (SREBP), a master regulator of mammalian lipogenesis, led to the discovery of a conjugate of docosahexaenoic acid (DHA), glucosamine, and amino acids as an inhibitor of SREBP (molecule 1, DHG). Mechanistic analyses indicate that molecule 1 impairs the SREBP activity by inhibiting glucose transporters and thereby activating AMP-activated protein kinase (AMPK). Oral administration of molecule 1 suppressed the intestinal absorption of glucose in mice. These results suggest that such synthetic libraries of nutrient conjugates serve as a source of novel chemical tools and pharmaceutical seeds that modulate energy metabolism.
Asunto(s)
Nutrientes/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidores , Aminoácidos/síntesis química , Aminoácidos/farmacología , Animales , Células CACO-2 , Ácidos Grasos Insaturados/síntesis química , Ácidos Grasos Insaturados/farmacología , Genes Reporteros , Glucosamina/síntesis química , Glucosamina/farmacología , Glucosa/metabolismo , Humanos , Absorción Intestinal/efectos de los fármacos , Masculino , Ratones Endogámicos ICR , Nutrientes/síntesis química , Bibliotecas de Moléculas Pequeñas/síntesis química , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Vitaminas/síntesis química , Vitaminas/farmacologíaRESUMEN
Structure-based design and synthesis of two biphenyl-N-acyl-ß-d-glucopyranosylamine derivatives as well as their assessment as inhibitors of human liver glycogen phosphorylase (hlGPa, a pharmaceutical target for type 2 diabetes) is presented. X-ray crystallography revealed the importance of structural water molecules and that the inhibitory efficacy correlates with the degree of disturbance caused by the inhibitor binding to a loop crucial for the catalytic mechanism. The in silico-derived models of the binding mode generated during the design process corresponded very well with the crystallographic data.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Glucosamina/análogos & derivados , Glucógeno Fosforilasa/química , Relación Estructura-Actividad Cuantitativa , Sitios de Unión , Dominio Catalítico , Técnicas de Química Sintética , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Glucosamina/síntesis química , Glucosamina/química , Glucosamina/farmacología , Glucógeno Fosforilasa/antagonistas & inhibidores , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Unión ProteicaRESUMEN
N-Azidoacetyl-d-glucosamine (GlcNAz) is a particularly useful tool in chemical biology as the azide is a metabolically stable yet accessible handle within biological systems. Herein, we report a practical synthesis of FmocAsn(N-Ac3GlcNAz)OH, a building block for solid phase peptide synthesis (SPPS). Protecting group manipulations are minimised by taking advantage of the inherent chemoselectivity of phosphine-mediated azide reduction, and the resulting glycosyl amine is employed directly in the opening of Fmoc protected aspartic anhydride. We show potential application of the building block by establishing it as a substrate for enzymatic glycan extension using sugar oxazolines of varying size and biological significance with several endo-ß-N-acetylglucosaminidases (ENGases). The added steric bulk resulting from incorporation of the azide is shown to have no or a minor impact on the yield of enzymatic glycan extension.
Asunto(s)
Asparagina/síntesis química , Asparagina/metabolismo , Glucosamina/síntesis química , Polisacáridos/química , Acetilglucosaminidasa/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Azidas/química , Glucosamina/metabolismo , Estructura Molecular , Oxidación-Reducción , Técnicas de Síntesis en Fase Sólida/métodos , Relación Estructura-ActividadRESUMEN
In the quest for discovering potent antimicrobial agents with lower toxicity, we envisioned the design and synthesis of nalidixic acid-D-(+)-glucosamine conjugates. The novel compounds were synthesized and evaluated for their in vitro antimicrobial activity against Gram positive bacteria, Gram negative bacteria and fungi. Cytotoxicity using MTT assay over L6 skeletal myoblast cell line, ATCC CRL-1458 was carried out. In vitro antimicrobial assay revealed that 1-ethyl-7-methyl-4-oxo-N-(1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose-2-yl)-[1,8]-naphthyridine-3-carboxamide (5) and 1-ethyl-7-methyl-4-oxo-N-(2-deoxy-D-glucopyranose-2-yl)-[1,8]-naphthyridine-3-carboxamide(6) possess growth inhibitory activity against resistant Escherichia coli NCTC, 11954 (MIC 0.1589 mM) and Methicillin resistant Staphylococcus aureus ATCC, 33591 (MIC 0.1589 mM). Compound (5) was more active against Listeria monocytogenes ATCC 19115 (MIC 0.1113 mM) in comparison with the reference nalidixic acid (MIC 1.0765 mM). Interestingly, compound (6) had potential antifungal activity against Candida albicans ATCC 10231 (MIC <0.0099 mM). Remarkably, the tested compounds had low cytotoxic effect. This study indicated that glucosamine moiety inclusion into the chemical structure of the marketed nalidixic acid enhances antimicrobial activity and safety.
Asunto(s)
Antiinfecciosos/síntesis química , Diseño de Fármacos , Glucosamina/síntesis química , Naftiridinas/síntesis química , Antiinfecciosos/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Glucosamina/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/fisiología , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mioblastos/efectos de los fármacos , Mioblastos/fisiología , Naftiridinas/farmacologíaRESUMEN
A glucosamine modified near-infrared cyanine dye CyT sensitive to pH was synthesized. Due to the different pH values of mitochondria and autolysosomes, the probe can simultaneously investigate mitochondria and autolysosomes in living cells. Moreover, due to the introduction of glucosamine groups, this fluorescent probe can be applied for tumor targeted imaging.
Asunto(s)
Colorantes Fluorescentes/farmacología , Glucosamina/análogos & derivados , Glucosamina/farmacología , Indoles/farmacología , Mitofagia , Neoplasias/diagnóstico por imagen , Animales , Línea Celular Tumoral , Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Glucosamina/síntesis química , Glucosamina/química , Humanos , Concentración de Iones de Hidrógeno , Indoles/síntesis química , Indoles/química , Lisosomas/metabolismo , Ratones , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Mitocondrias/metabolismo , Mitocondrias/fisiologíaRESUMEN
AIM: To design colon-targeted codrugs of mycophenolic acid (MPA) and aminosugars as a safer option to mycophenolate mofetil (MMF) in the management of inflammatory bowel disease. METHODS: Codrugs were synthesized by coupling MPA with aminosugars (D-glucosamine and D-galactosamine) using EDCI coupling. The structures were confirmed by infrared radiation, nuclear magnetic resonance, mass spectroscopy and elemental analysis. The release profile of codrugs was extensively studied in aqueous buffers, upper gastrointestinal homogenates, faecal matter and caecal homogenates (in vitro) and rat blood (in vitro). Anti-colitic activity was assessed in 2,4,6-trinitrobezenesulfonic acid-induced colitis in Wistar rats by the estimation of various demarcating parameters. Statistical evaluation was performed by applying one-way and two-way ANOVA when compared with the disease control. RESULTS: The prodrugs resisted activation in HCl buffer (pH 1.2) and stomach homogenates of rats with negligible hydrolysis in phosphate buffer (pH 7.4) and intestinal homogenates. Incubation with colon homogenates (in vitro) produced 76% to 89% release of MPA emphasizing colon-specific activation of codrugs and the release of MPA and aminosugars at the site of action. In the in vitro studies, the prodrug of MPA with D-glucosamine (MGLS) was selected which resulted in 68% release of MPA in blood. in vitro studies on MGLS revealed its colon-specific activation after a lag time of 8 h which could be ascribed to the hydrolytic action of N-acyl amidases found in the colon. The synthesized codrugs markedly diminished disease activity score and revived the disrupted architecture of the colon that was comparable to MMF but superior to MPA. CONCLUSION: The significant attenuating effect of prodrugs and individual aminosugars on colonic inflammation proved that the rationale of the codrug approach is valid.
Asunto(s)
Colitis Ulcerosa/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Profármacos/uso terapéutico , Animales , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/patología , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Modelos Animales de Enfermedad , Liberación de Fármacos , Sinergismo Farmacológico , Galactosamina/síntesis química , Galactosamina/farmacología , Galactosamina/uso terapéutico , Mucosa Gástrica/metabolismo , Glucosamina/síntesis química , Glucosamina/farmacología , Glucosamina/uso terapéutico , Humanos , Inmunosupresores/síntesis química , Inmunosupresores/farmacología , Masculino , Ácido Micofenólico/síntesis química , Ácido Micofenólico/farmacología , Ácido Micofenólico/uso terapéutico , Profármacos/síntesis química , Profármacos/farmacología , Ratas , Ratas Wistar , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Ácido Trinitrobencenosulfónico/toxicidadRESUMEN
Aryl substituted 1-(ß-d-glucosaminyl)-1,2,3-triazoles as well as C-ß-d-glucosaminyl 1,2,4-triazoles and imidazoles were synthesized and tested as inhibitors against muscle and liver isoforms of glycogen phosphorylase (GP). While the N-ß-d-glucosaminyl 1,2,3-triazoles showed weak or no inhibition, the C-ß-d-glucosaminyl derivatives had potent activity, and the best inhibitor was the 2-(ß-d-glucosaminyl)-4(5)-(2-naphthyl)-imidazole with a Ki value of 143 nM against human liver GPa. An X-ray crystallography study of the rabbit muscle GPb inhibitor complexes revealed structural features of the strong binding and offered an explanation for the differences in inhibitory potency between glucosyl and glucosaminyl derivatives and also for the differences between imidazole and 1,2,4-triazole analogues.
Asunto(s)
Glucosamina/análogos & derivados , Glucógeno Fosforilasa/antagonistas & inhibidores , Imidazoles/farmacología , Triazoles/farmacología , Animales , Cristalografía por Rayos X , Glucosamina/síntesis química , Glucosamina/farmacología , Humanos , Enlace de Hidrógeno , Imidazoles/síntesis química , Cinética , Hígado/enzimología , Músculo Esquelético/enzimología , Dominios Proteicos , Conejos , Relación Estructura-Actividad , Triazoles/síntesis químicaRESUMEN
Biofouling, an undesirable accumulation of organisms on sea-immersed structures such as ship hulls and fishing nets, is a serious economic issue whose effects include oil wastage and clogged nets. Organotin compounds were utilized since the 1960s as an antifouling material; however, the use of such compounds was later banned by the International Maritime Organization (IMO) due to their high toxicity toward marine organisms, resulting in masculinization and imposex. Since the ban, there have been extensive efforts to develop environmentally benign antifoulants. Natural antifouling products obtained from marine creatures have been the subject of considerable attention due to their potent antifouling activity and low toxicity. These antifouling compounds often contain isocyano groups, which are well known to have natural antifouling properties. On the basis of our previous total synthesis of natural isocyanoterpenoids, we envisaged the installation of an isocyano functional group onto glucosamine to produce an environmentally friendly antifouling material. This paper describes an effective synthetic method for various glucosamine-based isocyanides and evaluation of their antifouling activity and toxicity against cypris larvae of the barnacle Amphibalanus amphitrite. Glucosamine isocyanides with an ether functionality at the anomeric position exhibited potent antifouling activity, with EC50 values below 1 µg/mL, without detectable toxicity even at a high concentration of 10 µg/mL. Two isocyanides had EC50 values of 0.23 and 0.25 µg/mL, comparable to that of CuSO4, which is used as a fouling inhibitor (EC50 = 0.27 µg/mL).
Asunto(s)
Incrustaciones Biológicas/prevención & control , Cianuros/farmacología , Glucosamina/farmacología , Thoracica/efectos de los fármacos , Animales , Productos Biológicos/síntesis química , Productos Biológicos/química , Productos Biológicos/farmacología , Sulfato de Cobre/farmacología , Cianuros/síntesis química , Cianuros/química , Glucosamina/síntesis química , Glucosamina/química , Larva/efectos de los fármacosRESUMEN
6-Sulfo-6-deoxy-D-glucosamine (GlcN6S), 6-sulfo-6-deoxy-D-glucosaminitol (ADGS) and their N-acetyl and methyl ester derivatives have been synthesized and tested as inhibitors of enzymes catalyzing reactions of the UDP-GlcNAc pathway in bacteria and yeasts. GlcN6S and ADGS at micromolar concentrations inhibited glucosamine-6-phosphate (GlcN6P) synthase of microbial origin. The former was also inhibitory towards fungal GlcN6P N-acetyl transferase, but at millimolar concentrations. Both compounds and their N-acetyl derivatives exhibited antimicrobial in vitro activity, with MICs in the 0.125-2.0 mg mL-1 range. Antibacterial but not antifungal activity of GlcN6S was potentiated by D-glucosamine and a synergistic antibacterial effect was observed for combination of ADGP and a dipeptide Nva-FMDP.
Asunto(s)
Antiinfecciosos/síntesis química , Antiinfecciosos/farmacología , Glucosamina/síntesis química , Glucosamina/farmacología , Tioazúcares/farmacología , Antiinfecciosos/química , Antiinfecciosos/metabolismo , Técnicas de Química Sintética , Glucosamina/química , Glucosamina/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Espacio Intracelular/metabolismo , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Conformación Proteica , Tioazúcares/síntesis química , Tioazúcares/química , Tioazúcares/metabolismoRESUMEN
Insulin-secreting beta cells together with glucagon-producing alpha cells play an essential role in maintaining the optimal blood glucose level in the body, so the development of selective probes for imaging of these cell types in live islets is highly desired. Herein we report the development of a 2-glucosamine-based two-photon fluorescent probe, TP-ß, that is suitable for imaging of beta cells in live pancreatic islets from mice. Flow cytometry studies confirmed that TP-ß is suitable for isolation of primary beta cells. Moreover, two-photon imaging of TP-ß-stained pancreatic islets showed brightly stained beta cells in live islets. Insulin enzyme-linked immunosorbent assays revealed that TP-ß has no effect on glucose-stimulated insulin secretion from the stained islet. Finally, to develop a more convenient islet imaging application, we combined our recently published alpha-cell-selective probe TP-α with TP-ß to make a "TP islet cocktail". This unique dye cocktail enabled single excitation (820 nm) and simultaneous dual-color imaging of alpha cells (green) and beta cells (red) in live pancreatic islets. This robust TP islet cocktail may serve as a valuable tool for basic diabetic studies.
Asunto(s)
Colorantes Fluorescentes/química , Glucosamina/química , Imagenología Tridimensional , Células Secretoras de Insulina/citología , Fotones , Supervivencia Celular , Células Cultivadas , Colorantes Fluorescentes/síntesis química , Glucosamina/síntesis química , Humanos , Estructura MolecularRESUMEN
Bacteria communicate with one another and regulate their pathogenicity through a phenomenon known as quorum sensing (QS). When the bacterial colony reaches a threshold density, the QS system induces the production of virulence factors and the formation of biofilms, a powerful defence system against the host's immune responses. The glucosamine monomer has been shown to disrupt the bacterial QS system by inhibiting autoinducer (AI) signalling molecules such as the acyl-homoserine lactones (AHLs). In this study, the synthesis of acetoxy-glucosamides 8, hydroxy-glucosamides 9 and 3-oxo-glucosamides 12 was performed via the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N,N'-dicyclohexylcarbodiimide (DCC) coupling methods. All of the synthesized compounds were tested against two bacterial strains, P. aeruginosa MH602 (LasI/R-type QS) and E. coli MT102 (LuxI/R-type QS), for QS inhibitory activity. The most active compound 9b showed 79.1% QS inhibition against P. aeruginosa MH602 and 98.4% against E. coli MT102, while compound 12b showed 64.5% inhibition against P. aeruginosa MH602 and 88.1% against E. coli MT102 strain at 2mM concentration. The ability of the compounds to inhibit the production of the virulence factor pyocyanin and biofilm formation in the P. aeruginosa (PA14) strain was also examined. Finally, computational docking studies were performed with the LasR receptor protein.
Asunto(s)
Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Glucosamina/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Glucosamina/síntesis química , Glucosamina/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
A series of five new fluorescent deoxynojirimycin (DNJ) conjugates were synthesized and evaluated for their inhibitory effect (IC50) on several α- and ß-glucosidases. Three of the conjugates showed enhanced activity. The two synthetic conjugates, DNJ-CF31 and DNJ-Me 2, exhibited improved α-glucosidase inhibitory effects compared to DNJ and miglitol. Interestingly, conjugates 1 and 2 showed strong inhibition of almond-derived ß-glucosidase, in contrast to the inhibition tendencies of other inhibitors. Conjugate 5 strongly inhibited rat intestinal maltase, even at 0.10µM. A docking study indicated that all five conjugates bind to the active site of α-glucosidase (PDB: 3L4V, derived from Homo sapiens). The DNJ portion of the conjugate fits into the cavity of the enzyme, and the fluorescent part locates randomly on the outside surface. Thus, it is likely that these conjugates can specifically recognize intestinal cells, specifically the α-glucosidase on cell membranes.
Asunto(s)
Colorantes Fluorescentes/química , Glucosamina/análogos & derivados , Inhibidores de Glicósido Hidrolasas/síntesis química , Inhibidores de Glicósido Hidrolasas/farmacología , alfa-Glucosidasas/metabolismo , 1-Desoxinojirimicina/síntesis química , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes/síntesis química , Glucosamina/síntesis química , Glucosamina/química , Glucosamina/farmacología , Inhibidores de Glicósido Hidrolasas/química , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via a 1,2-anhydrosugar "epoxide" mechanism that proceeds through an unusual conformational itinerary. Here, we explore how shape and charge contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-α-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how the neutral inhibitors provide shape but not charge mimicry of the proposed intermediate and transition state structures. Building upon the knowledge of shape and charge contributions to inhibition of family GH99 enzymes, we design and synthesize α-Man-1,3-noeuromycin, which is revealed to be the most potent inhibitor (KD 13 nM for Bacteroides xylanisolvens GH99 enzyme) of these enzymes yet reported. This work reveals how shape and charge mimicry of transition state features can enable the rational design of potent inhibitors.
Asunto(s)
Glucosamina/análogos & derivados , Inhibidores de Glicósido Hidrolasas/farmacología , Manosidasas/antagonistas & inhibidores , Conformación de Carbohidratos , Cristalografía por Rayos X , Glucosamina/síntesis química , Glucosamina/química , Glucosamina/farmacología , Inhibidores de Glicósido Hidrolasas/síntesis química , Inhibidores de Glicósido Hidrolasas/química , Manosidasas/metabolismo , Modelos MolecularesRESUMEN
In this communication, we describe a three-step synthesis of l-ido-1-deoxynojirimycin derivatives starting from readily available 2,3,4,6-tetra-O-benzyl-d-glucopyranose via Ir-catalyzed reductive amination in water, "borrowing hydrogen" under neat conditions, and Pd-catalyzed debenzylation.