RESUMEN
A collection of new reversible glycosidase inhibitors of the iminoalditol type featuring N-substituents containing perfluorinated regions has been prepared for evaluation of physicochemical, biochemical and diagnostic properties. The vast variety of feasible oligofluoro moieties allows for modular approaches to customised structures according to the intended applications, which are influenced by the fluorine content as well as the distance of the fluorous moiety from the ring nitrogen. The first examples, in particular in the D-galacto series, exhibited excellent inhibitory activities. A preliminary screen with two human cell lines showed that, at subinhibitory concentrations, they are powerful pharmacological chaperones enhancing the activities of the catalytically handicapped lysosomal D-galactosidase mutants associated with GM1 gangliosidosis and Morquio B disease.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Galactosidasas/antagonistas & inhibidores , Gangliosidosis GM1/tratamiento farmacológico , Alcoholes del Azúcar/química , Alcoholes del Azúcar/farmacología , Línea Celular , Café/enzimología , Inhibidores Enzimáticos/uso terapéutico , Escherichia coli/enzimología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Galactosidasas/metabolismo , Halogenación , Humanos , Iminas/química , Iminas/farmacología , Iminas/uso terapéutico , Rhizobium/enzimología , Alcoholes del Azúcar/uso terapéuticoRESUMEN
Biotin-iminosugar conjugates of different configuration such as D-gluco, D-galacto, L-ido as well as a furanoid representative in the D-manno configuration have been synthesised and exhibit powerful inhibition of beta-glucosidase from Agrobacterium sp. with K(i) values in the range of the respective parent compounds. Such molecular probes have potential for activity-based protein profiling taking advantage of the biotin-(strept)avidin interaction.
Asunto(s)
Biotina/síntesis química , Biotina/farmacología , Alcoholes del Azúcar/síntesis química , Alcoholes del Azúcar/farmacología , Evaluación Preclínica de Medicamentos , Sondas MolecularesRESUMEN
N-Alkylation at the ring nitrogen of the D-galactosidase inhibitor 1-deoxygalactonojirimycin with a functionalised C 6alkyl chain followed by modification with different aromatic substituents provided lipophilic 1-deoxygalactonojirimycin derivatives which exhibit inhibitory properties against ß-glycosidases from E. coli and Agrobacterium sp. as well as green coffee bean α-galactosidase. In preliminary studies, these compounds also showed potential as chemical chaperones for GM1-gangliosidosis related ß-galactosidase mutants.
RESUMEN
The N-glycosylation pathway is a target for pharmaceutical intervention in a number of pathological conditions including cancer. Golgi alpha-mannosidase II (GMII) is the final glycoside hydrolase in the pathway and has been the target for a number of synthetic efforts aimed at providing more selective and effective inhibitors. Drosophila GMII (dGMII) has been extensively studied due to the ease of obtaining high resolution structural data, allowing the observation of substrate distortion upon binding and after formation of a trapped covalent reaction intermediate. However, attempts to find new inhibitor leads by high-throughput screening of large commercial libraries or through in silico docking were unsuccessful. In this paper we provide a kinetic and structural analysis of five inhibitors derived from a small glycosidase-focused library. Surprisingly, four of these were known inhibitors of beta-glucosidases. X-ray crystallographic analysis of the dGMII:inhibitor complexes highlights the ability of the zinc-containing GMII active site to deform compounds, even ones designed as conformationally restricted transition-state mimics of beta-glucosidases, into binding entities that have inhibitory activity. Although these deformed conformations do not appear to be on the expected conformational itinerary of the enzyme, and are thus not transition-state mimics of GMII, they allow positioning of the three vicinal hydroxyls of the bound gluco-inhibitors into similar locations to those found with mannose-containing substrates, underlining the importance of these hydrogen bonds for binding. Further, these studies show the utility of targeting the acid-base catalyst using appropriately positioned positively charged nitrogen atoms, as well as the challenges associated with aglycon substitutions.
Asunto(s)
Inhibidores Enzimáticos/química , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/química , Aparato de Golgi/enzimología , Manosidasas/antagonistas & inhibidores , Manosidasas/química , Animales , Células Cultivadas , Cristalografía por Rayos X , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/enzimología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/farmacología , Aparato de Golgi/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína/fisiología , Proteínas/antagonistas & inhibidores , Proteínas/químicaRESUMEN
Specific inhibitors of human pancreatic alpha-amylase (HPA) have potential as oral agents for the control of blood glucose levels in the treatment of diabetes and obesity. In a search for novel inhibitors, a library of 30 000 crude biological extracts of terrestrial and marine origin has been screened. A number of inhibitory extracts were identified, of which the most potent was subjected to bioassay-guided purification. A family of three glycosylated acyl flavonols, montbretins A-C, was thereby identified and characterized as competitive amylase inhibitors, with K(i) values ranging from 8.1-6100 nM. Competitive inhibition by myricetin, which corresponds to the flavone core, and noncompetitive inhibition by a second fragment, ethyl caffeiate, suggest a binding mode for these inhibitors.
Asunto(s)
Productos Biológicos/farmacología , Inhibidores Enzimáticos/farmacología , Páncreas/enzimología , alfa-Amilasas/antagonistas & inhibidores , Glicosilación , HumanosRESUMEN
A high-throughput screen to detect the synthesis of natural and non-natural gangliosides by cell lysates has been developed and automated. Utilizing the binding specificity of cholera toxin B-subunit for the oligosaccharide moiety of the ganglioside G M1, the synthesis of sugar-sphingolipid glycosidic linkages was detected using a modified enzyme-linked immunosorbent assay (ELISA)/enzyme-linked lectin assay (ELLA). The screen was optimized and validated for high-throughput screening of cell lysates by evaluating different vectors, promoters, substrates and detection strategies. The extent of ganglioside synthesis was found to be proportional to enzyme concentration and length of incubation time. As a test of the finalized screen efficacy, individual colonies from a saturation mutagenesis library of nucleophile mutants of an endoglycoceramidase were screened to identify the most active enzyme for ganglioside synthesis. This screen should find general application in assaying both glycolipid biosynthesis and glycolipid hydrolysis, as it is highly sensitive and can be used with crude cell extracts.
Asunto(s)
Extractos Celulares/química , Gangliósidos/biosíntesis , Animales , Anticuerpos/metabolismo , Biotina/metabolismo , Catálisis , Bovinos , Extractos Celulares/análisis , Toxina del Cólera/metabolismo , Fluoruros/química , Fluoruros/metabolismo , Glicósido Hidrolasas/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Ratones , Esfingosina/metabolismo , Estreptavidina/metabolismoRESUMEN
Three typical glycosidase-inhibiting iminoalditols were attached to a polyamine surface displayed on a silicon chip. Exposure to a representative beta-glucosidase revealed selective binding events reflecting the different structural features of the inhibitors probed in this study. This provides a proof-of-concept for the successful exploitation of microarrays of typical reversible glycosidase inhibitors of the iminosugar family.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/metabolismo , Iminoazúcares/química , Iminoazúcares/farmacología , Análisis por Micromatrices , Espectroscopía de Resonancia Magnética , Estructura Molecular , Rhizobium/enzimologíaRESUMEN
Cyclization by double reductive amination of L-arabino-hexos-5-ulose with suitably protected D- as well as L-lysine derivatives provided 1-deoxygalactonojirimycin lysine hybrids without any observable epimer formation at C-5. Modifications on the lysine moiety by acylation gave access to lipophilic derivatives which exhibited excellent D-galactosidase inhibitory activities.
Asunto(s)
1-Desoxinojirimicina/química , Inhibidores Enzimáticos/síntesis química , Galactosidasas/antagonistas & inhibidores , Lisina/química , Acilación , Quimera , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Galactosidasas/metabolismo , CinéticaRESUMEN
The Golgi-resident glycosyltransferase, UDP-N-acetyl-d-glucosamine:alpha-3-d-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I), initiates the conversion of high-mannose oligosaccharides to complex and hybrid structures in the biosynthesis of N-linked glycans. Reported here are the X-ray crystal structures of GnT I in complex with UDP-CH2-GlcNAc (a non-hydrolyzable C-glycosidic phosphonate), UDP-2-deoxy-2-fluoro-glucose, UDP-glucose and UDP. Collectively, these structures provide evidence for the importance of the GlcNAc moiety and its N-acetyl group in donor substrate binding, as well as insight into the role played by the flexible 318-330 loop in substrate binding and product release. In addition, the UDP-CH2-GlcNAc complex reveals a well-defined glycerol molecule poised for nucleophilic attack on the C1 atom of the donor substrate analogue. The position and orientation of this glycerol molecule have allowed us to model the binding of the Manalpha1,3Manbeta1 moiety of the acceptor substrate and, based on the model, to suggest a rationalization for the main determinants of GnT I acceptor specificity.
Asunto(s)
N-Acetilglucosaminiltransferasas/química , Animales , Catálisis , Cristalografía por Rayos X , Glicosiltransferasas/química , Cinética , Manosa/química , Modelos Químicos , Modelos Moleculares , Oligosacáridos/química , Unión Proteica , Estructura Terciaria de Proteína , Conejos , Especificidad por SustratoRESUMEN
Cyclization by double reductive amination of D-xylo-hexos-5-ulose with the terminal amino group of alpha-N-Boc-lysine methyl ester gave a 4:1-mixture of (1'R)-N-methoxycarbonyl-(1-N-Boc-amino)pentyl-1-deoxynojirimycin and the corresponding L-ido epimer whereas D-lyxo-hexos-5-ulose furnished the desired N-alkylated 1-deoxymannojirimycin derivative without any observable epimer formation at C-5. By subsequent modification of the lysine moiety, additional chain-extended derivatives as well as fluorescent compounds were obtained. All fluorescent iminoalditol-amino acid hybrids prepared in this study exhibited glycosidase inhibitory activities better than or comparable to the parent compounds'.
Asunto(s)
Aminoácidos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glicósido Hidrolasas/antagonistas & inhibidores , Iminas , Alcoholes del Azúcar/química , Aminoácidos/farmacología , Inhibidores Enzimáticos/química , Glucosidasas/antagonistas & inhibidores , Indicadores y Reactivos , Cinética , Manosidasas/antagonistas & inhibidores , Modelos Moleculares , Alcoholes del Azúcar/síntesis química , Alcoholes del Azúcar/farmacologíaRESUMEN
Based on our extensive studies of D-glucosidase inhibiting 2,5-dideoxy-2,5-imino-D-mannitol derivatives, we have been trying to create a series of fluorescent derivatives with a view to an 'inhibitory activity ruler' based on competitive displacement reactions of non-fluorescent inhibitors by fluorescent ones and vice versa, which can be performed and followed in microtiter plates or on-chips. Thus, a set of compounds was assembled with Ki values between 2 nM and 1 microM against Agrobacterium sp. beta-glucosidase.
Asunto(s)
Manitol/análogos & derivados , beta-Glucosidasa/antagonistas & inhibidores , Iminopiranosas/farmacología , Cinética , Manitol/farmacología , Rhizobium/enzimología , Relación Estructura-ActividadRESUMEN
The formation of glycoconjugates and oligosaccharides remains one of the most challenging chemical syntheses. Chemo-enzymatic routes using retaining glycosidases have been successfully harnessed but require tight kinetic or thermodynamic control. "Glycosynthases," specifically engineered glycosidases that catalyze the formation of glycosidic bonds from glycosyl donor and acceptor alcohol, are an emerging range of synthetic tools in which catalytic nucleophile mutants are harnessed together with glycosyl fluoride donors to generate powerful and versatile catalysts. Here we present the structural and kinetic dissection of the Humicola insolens Cel7B glycosynthases in which the nucleophile of the wild-type enzyme is mutated to alanine and serine (E197A and E197S). 3-D structures reveal the acceptor and donor subsites and the basis for substrate inhibition. Kinetic analysis shows that the E197S mutant is considerably more active than the corresponding alanine mutant due to a 40-fold increase in k(cat).
Asunto(s)
Hongos/metabolismo , Glicósido Hidrolasas/metabolismo , Glicósidos/biosíntesis , Lactosa/análogos & derivados , Celobiosa/metabolismo , Cristalización , Interpretación Estadística de Datos , Hongos/enzimología , Indicadores y Reactivos , Cinética , Modelos Moleculares , Mutagénesis/genética , Oligosacáridos/metabolismoRESUMEN
Immobilized biocatalysts, including particulate enzymes, represent an attractive tool for research and industrial applications because they combine the specificity of native enzymes with the advantage that they can be readily separated from end product and reused. We demonstrated the use of the Caulobacter crescentus surface (S)-layer protein (RsaA) secretion apparatus for the generation of particulate enzymes. Specifically, a candidate protein made previously by fusion of the beta-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi with the C-terminus of RsaA was evaluated. Cex/RsaA cleaved the glycosidic linkage in the artificial substrate p-nitrophenyl-beta-D-cellobioside with a KM similar to that of native Cex (1.1 mM for Cex/RsaA vs 0.60 mM for Cex), indicating that the particulate Cex enzyme was able to bind substrate with wild-type affinity. By contrast, the kcat value was significantly reduced (0.08 s-1 for Cex/RsaA vs 15.8 s-1 for Cex), likely owing to the fact that the RsaA C-terminus induces spontaneous unstructured aggregation of the recombinant protein. Here, we demonstrated that not only can an RsaA fusion protein be cheaply produced and purified to a high yield (76 mg/L of dry wt for Cex/RsaA), but it can also be efficiently recycled. The Caulobacter S-layer secretion system therefore offers an attractive new model system for the production of particulate biocatalysts.
Asunto(s)
Proteínas Bacterianas/química , Caulobacter crescentus/enzimología , Cellulomonas/enzimología , Enzimas Inmovilizadas , Glicósido Hidrolasas/química , Glicoproteínas de Membrana/química , Proteínas Recombinantes de Fusión , Estabilidad de Enzimas , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/aislamiento & purificación , Cinética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificaciónAsunto(s)
Quitinasas/antagonistas & inhibidores , Disacáridos , Tiazolidinas , Trisacáridos , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Disacáridos/química , Disacáridos/metabolismo , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Serratia marcescens/enzimología , Tiazolidinas/química , Tiazolidinas/metabolismo , Trisacáridos/química , Trisacáridos/metabolismoRESUMEN
The atomic-resolution structure of a xylobiose-derived isofagomine lactam in complex with the xylanase Xyn10A from Streptomyces lividans reveals that the lactam is bound to the enzyme as the amide tautomer, with "reversed" protonation-states for nucleophile and acid-base.
Asunto(s)
Amidas/química , Disacáridos/química , Inhibidores Enzimáticos/química , Glicósido Hidrolasas/antagonistas & inhibidores , Lactamas/química , Piperidinas/química , Unión Competitiva , Cristalografía por Rayos X , Disacáridos/metabolismo , Disacáridos/farmacología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Glicósido Hidrolasas/metabolismo , Iminopiranosas , Lactamas/metabolismo , Lactamas/farmacología , Modelos Moleculares , Piperidinas/metabolismo , Piperidinas/farmacología , Streptomyces/enzimología , Xilano Endo-1,3-beta-Xilosidasa , Xilosidasas/metabolismoRESUMEN
The atomic resolution structures of xylobiose-derived isofagomine and xylobiose-derived deoxynojirimycin in complex with the xylanase Xyn10A from Streptomyces lividans reveal undistorted (4)C(1) chair conformed sugars and, in the case of the deoxynojirimycin analogue, suggest unusual pK(a) changes of the enzyme's catalytic machinery upon binding.
Asunto(s)
1-Desoxinojirimicina/química , Disacáridos/química , Piperidinas/química , Streptomyces lividans/enzimología , Xilosidasas/química , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Iminopiranosas , Espectrofotometría Atómica/métodosRESUMEN
A series of mannotrisaccharides were synthesized by two distinct chemical pathways as probes of the enzymes involved in the early stages of mammalian complex N-glycan formation. Methyl (alpha-D-mannopyranosyl)-(1-->3)-[(alpha-D-mannopyranosyl)-(1-->6)]-beta-D-mannopyranoside (6) and methyl (2-deoxy-2-fluoro-alpha-D-mannopyranosyl)-(1-->3)-[(2-deoxy-2-fluoro-alpha-D-mannopyranosyl)-(1-->6)]-beta-D-mannopyranoside (8) were rapidly synthesized from unprotected methyl beta-D-mannopyranoside (12). Methyl (2-deoxy-2-fluoro-alpha-D-mannopyranosyl)-(1-->3)-[(alpha-D-mannopyranosyl)-(1-->6)]-beta-D-mannopyranoside (7) and methyl (alpha-D-mannopyranosyl)-(1-->3)-[(2-deoxy-2-fluoro-alpha-D-mannopyranosyl)-(1-->6)]-beta-D-mannopyranoside (9) were synthesized from the common orthogonally protected precursor methyl 2-O-acetyl-4,6-O-benzylidene-beta-D-mannopyranoside (15). The 2-deoxy-2-fluoro substitution common to trisaccharides 7-9 renders these analogues resistant to enzyme action in two distinct ways. Firstly the fluorine serves as a non-nucleophilic isostere for the acceptor hydroxyl in studies with glycosyl transferases GnT-I and GnT-II (7 and 9, respectively). Secondly it should render trisaccharide 8 stable to hydrolysis by the mannosidases Man-II and Man-III by inductive destabilization of their oxocarbenium ion-like transition states. These analogues should be useful for structural studies on these enzymes.
Asunto(s)
Enzimas/química , Sondas Moleculares/síntesis química , Polisacáridos/biosíntesis , Trisacáridos/síntesis química , Conformación de Carbohidratos , Inhibidores Enzimáticos/síntesis química , Glicosiltransferasas/antagonistas & inhibidores , Glicosiltransferasas/química , Manosa , Manosidasas/antagonistas & inhibidores , Manosidasas/química , Sondas Moleculares/química , Trisacáridos/químicaRESUMEN
Cyclisation by double reductive amination of 2-acetamino-2-deoxy-D-xylo-hexos-5-ulose with N-2 protected L-lysine derivatives provided 2-acetamino-1,2-dideoxynojirimycin derivatives without any observable epimer formation at C-5. Modifications on the lysine moiety gave access to lipophilic derivatives that exhibited improved hexosaminidase inhibitory activities.
RESUMEN
The contributions of enzyme-substrate hydrogen-binding interactions to catalysis by two different families of xylanases were evaluated through kinetic studies with two representative wild-type enzymes, Cellulomonas fimi xylanase (Cex) and Bacillus circulans xylanase (Bcx), on a series of monodeoxygenated and monodeoxyfluorinated p-nitrophenyl xylobioside substrates. Effects of substitution in the distal (-2 subsite) sugar on kcat/Km for Cex were moderately large (up to 2.9 kcal mol-1), with no effect seen on kcat. By contrast, substantial effects upon both kcat and kcat/Km were seen for substrates modified in the proximal (-1 subsite) sugar. Very similar results were obtained with Bcx. Kinetic analyses with a series of eight mutants of Cex in which active site residues interacting with the substrate were mutated yielded complementary insights. Again, interactions with the distal (-2) sugar were seen to contribute substantially to kcat/Km (up to 3.7 kcal mol-1), thus to the formation of the glycosyl-enzyme intermediate, but not to kcat, thus to the hydrolysis of the glycosyl-enzyme. Interactions with the proximal (-1) sugar are much more significant, contributing up to 6.7 kcal mol-1 to both kcat/Km and kcat. These results together indicate that interactions with the distal sugar maintain similar magnitudes in the transition states for glycosylation and deglycosylation as well as in the glycosyl-enzyme intermediate and can be referred to as "uniform binding interactions" in the parlance of Albery and Knowles (Albery, W. J., and Knowles, J. R. (1976) Biochemistry 15, 5631-5640). Interactions with the proximal sugar are considerably stronger at the deglycosylation transition state than in the intermediate, and fall into the category of differential binding interactions. This behavior likely has its origins in the changes in ring conformation of the proximal sugar but not of the distal sugar between the ground state and the reaction transition state. Correlation of these individual interaction energies with the hydrogen-bonding pattern seen in the glycosyl-enzyme intermediate allows for the assignment of hydrogen-bond strengths to each interaction, with good correlation between the two approaches. These findings are relevant to the discussion of remote binding effects upon enzymatic catalysis.