RESUMEN
Most enveloped viruses rely on the host cell endoplasmic reticulum (ER) quality control (QC) machinery for proper folding of glycoproteins. The key ER α-glucosidases (α-Glu) I and II of the ERQC machinery are attractive targets for developing broad-spectrum antivirals. Iminosugars based on deoxynojirimycin have been extensively studied as ER α-glucosidase inhibitors; however, other glycomimetic compounds are less established. Accordingly, we synthesized a series of N-substituted derivatives of valiolamine, the iminosugar scaffold of type 2 diabetes drug voglibose. To understand the basis for up to 100,000-fold improved inhibitory potency, we determined high-resolution crystal structures of mouse ER α-GluII in complex with valiolamine and 10 derivatives. The structures revealed extensive interactions with all four α-GluII subsites. We further showed that N-substituted valiolamines were active against dengue virus and SARS-CoV-2 in vitro. This study introduces valiolamine-based inhibitors of the ERQC machinery as candidates for developing potential broad-spectrum therapeutics against the existing and emerging viruses.
Asunto(s)
Antivirales/farmacología , Inhibidores de Glicósido Hidrolasas/farmacología , Iminoazúcares/farmacología , Inositol/análogos & derivados , alfa-Glucosidasas/metabolismo , Animales , Antivirales/síntesis química , Antivirales/metabolismo , Sitios de Unión , Chlorocebus aethiops , Cristalografía por Rayos X , Virus del Dengue/efectos de los fármacos , Retículo Endoplásmico/enzimología , Inhibidores de Glicósido Hidrolasas/síntesis química , Inhibidores de Glicósido Hidrolasas/metabolismo , Humanos , Iminoazúcares/síntesis química , Iminoazúcares/metabolismo , Inositol/síntesis química , Inositol/metabolismo , Inositol/farmacología , Ratones , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Unión Proteica , SARS-CoV-2/efectos de los fármacos , Células Vero , alfa-Glucosidasas/químicaRESUMEN
We have used the Cu(i)-catalyzed azide-alkyne Huisgen cycloaddition reaction to obtain two families of bivalent heterodimers where tacrine is connected to an azasugar or iminosugar, respectively, via linkers of variable length. The heterodimers were investigated as cholinesterase inhibitors and it was found that their activity increased with the length of the linker. Two of the heterodimers were significantly stronger acetylcholinesterase inhibitors than the monomeric tacrine. Molecular modelling indicated that the longer heterodimers fitted better into the active gorge of acetylcholinesterase than the shorter counterparts and the former provided more efficient simultaneous interaction with the tryptophan residues in the catalytic anionic binding site (CAS) and the peripheral anionic binding site (PAS).
Asunto(s)
Inhibidores de la Colinesterasa/química , Iminoazúcares/química , Tacrina/química , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Animales , Butirilcolinesterasa/química , Butirilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/metabolismo , Electrophorus , Pruebas de Enzimas , Caballos , Iminoazúcares/síntesis química , Iminoazúcares/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Unión Proteica , Relación Estructura-Actividad , Tacrina/síntesis química , Tacrina/metabolismo , TermodinámicaRESUMEN
Inhibition of the biosynthesis of complex N-glycans in the Golgi apparatus is one of alternative ways to suppress growth of tumor tissue. Eight N-benzyl substituted 1,4-imino-l-lyxitols with basic functional groups (amine, amidine, guanidine), hydroxyl and fluoro groups were prepared, optimized their syntheses and tested for their ability to inhibit several α-mannosides from the GH family 38 (GMIIb, LManII and JBMan) as models for human Golgi and lysosomal α-mannoside II. All compounds were found to be selective inhibitors of GMIIb. The most potent structure bearing guanidine group, inhibited GMIIb at the micromolar level (Kiâ¯=â¯19⯱â¯2⯵M) while no significant inhibition (>2â¯mM) of LManII and JBMan was observed. Based on molecular docking and pKa calculations this structure may form two salt bridges with aspartate dyad of the target enzyme improving its inhibitory potency compared with other N-benzyl substituted derivatives published in this and previous studies.
Asunto(s)
Inhibidores Enzimáticos/química , Iminoazúcares/química , Alcoholes del Azúcar/química , alfa-Manosidasa/antagonistas & inhibidores , Animales , Dominio Catalítico , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Pruebas de Enzimas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Iminoazúcares/síntesis química , Iminoazúcares/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Alcoholes del Azúcar/síntesis química , Alcoholes del Azúcar/metabolismo , alfa-Manosidasa/química , alfa-Manosidasa/metabolismoRESUMEN
Multivalent design of glycosidase inhibitors is a promising strategy for the treatment of diseases involving enzymatic hydrolysis of glycosidic bonds in carbohydrates. An essential prerequisite for successful applications is the atomic-level understanding of how outstanding binding enhancement occurs with multivalent inhibitors. Herein we report the first high-resolution crystal structures of the Jack bean α-mannosidase (JBα-man) in apo and inhibited states. The three-dimensional structure of JBα-man in complex with the multimeric cyclopeptoid-based inhibitor displaying the largest binding enhancements reported so far provides decisive insight into the molecular mechanisms underlying multivalent effects in glycosidase inhibition.
Asunto(s)
alfa-Manosidasa/metabolismo , Sitios de Unión , Canavalia/enzimología , Dominio Catalítico , Cristalografía por Rayos X , Iminoazúcares/química , Iminoazúcares/metabolismo , Estructura Terciaria de Proteína , Zinc/química , Zinc/metabolismo , alfa-Manosidasa/antagonistas & inhibidoresRESUMEN
Novel pyrrolidine-based multivalent iminosugars, synthesized by a CuAAC approach, have shown remarkable multivalent effects towards jack bean α-mannosidase and a Golgi α-mannosidase from Drosophila melanogaster, as well as a good selectivity with respect to a lysosomal α-mannosidase, which is important for anticancer applications. STD NMR and molecular modeling studies supported a multivalent mechanism with specific interactions of the bioactive iminosugars with Jack bean α-mannosidase. TEM studies suggested a binding mode that involves the formation of aggregates, which result from the intermolecular cross-linked network of interactions between the multivalent inhibitors and two or more dimers of JBMan heterodimeric subunits.
Asunto(s)
Pirrolidinas/metabolismo , alfa-Manosidasa/metabolismo , Animales , Sitios de Unión , Dominio Catalítico , Drosophila melanogaster/enzimología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Iminoazúcares/síntesis química , Iminoazúcares/química , Iminoazúcares/metabolismo , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Microscopía Electrónica de Transmisión , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Pirrolidinas/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , alfa-Manosidasa/antagonistas & inhibidores , alfa-Manosidasa/genéticaRESUMEN
The enigmatical dichotomy between the two CERT/GPBP protein isoforms, their vast panel of biological implications and the scarcity of known antagonist series call for new ligand chemotypes identification. We report the design of iminosugar-based ceramide mimics for the development of new START domain ligands potentially targeting either protein isoforms. Strategic choice of (i) an iminoxylitol core structure and (ii) the positioning of two dodecyl residues led to an extent of protein binding comparable to that of the natural cargo lipid ceramide or the archetypical inhibitor HPA-12. Molecular docking study evidenced a possible mode of protein binding fully coherent with the one observed in crystalline co-structures of known ligands. The present study thus paves the way for cellular CERT inhibition studies en route to the development of pharmacological tools aiming at deciphering the respective function and therapeutic potential of the two CERT/GPBP protein isoforms.
Asunto(s)
Iminoazúcares/química , Imitación Molecular , Isoformas de Proteínas/metabolismo , Espectroscopía de Resonancia Magnética con Carbono-13 , Diseño de Fármacos , Iminoazúcares/metabolismo , Ligandos , Simulación del Acoplamiento Molecular , Espectroscopía de Protones por Resonancia Magnética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
It has long been thought that iminosugar antiviral activity is a function of inhibition of endoplasmic reticulum-resident α-glucosidases, and on this basis, many iminosugars have been investigated as therapeutic agents for treatment of infection by a diverse spectrum of viruses, including dengue virus (DENV). However, iminosugars are glycomimetics possessing a nitrogen atom in place of the endocyclic oxygen atom, and the ubiquity of glycans in host metabolism suggests that multiple pathways can be targeted via iminosugar treatment. Successful treatment of patients with glycolipid processing defects using iminosugars highlights the clinical exploitation of iminosugar inhibition of enzymes other than ER α-glucosidases. Evidence correlating antiviral activity with successful inhibition of ER glucosidases together with the exclusion of alternative mechanisms of action of iminosugars in the context of DENV infection is limited. Celgosivir, a bicyclic iminosugar evaluated in phase Ib clinical trials as a therapeutic for the treatment of DENV infection, was confirmed to be antiviral in a lethal mouse model of antibody-enhanced DENV infection. In this study we provide the first evidence of the antiviral activity of celgosivir in primary human macrophages in vitro, in which it inhibits DENV secretion with an EC50 of 5 µM. We further demonstrate that monocyclic glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. By comparison to bicyclic glucose-mimicking iminosugars which inhibit glycoprotein processing but do not inhibit glycolipid processing and galactose-mimicking iminosugars which do not inhibit glycoprotein processing but do inhibit glycolipid processing, we demonstrate that inhibition of endoplasmic reticulum-resident α-glucosidases, not glycolipid processing, is responsible for iminosugar antiviral activity against DENV. Our data suggest that inhibition of ER α-glucosidases prevents release of virus and is the primary antiviral mechanism of action of iminosugars against DENV.
Asunto(s)
Antivirales/metabolismo , Virus del Dengue/crecimiento & desarrollo , Retículo Endoplásmico/enzimología , Inhibidores Enzimáticos/metabolismo , Iminoazúcares/metabolismo , Indolizinas/metabolismo , alfa-Glucosidasas/metabolismo , Animales , Células Cultivadas , Virus del Dengue/fisiología , Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Iminoazúcares/química , Indolizinas/química , Macrófagos/efectos de los fármacos , Macrófagos/virología , Modelos Moleculares , Estructura Molecular , Liberación del Virus/efectos de los fármacosRESUMEN
Several families of iminosugar-based galactoside mimics were designed, synthesized, and evaluated as galactocerebrosidase (GALC) inhibitors. They were also tested as inhibitors of lysosomal ß- and α-galactosidases in order to find new potent and selective pharmacological chaperones for treatment of the lysosomal storage disorder, Krabbe disease. Whereas 1-C-alkyl imino-L-arabinitols are totally inactive toward the three enzymes, 1-C-alkyl imino-D-galactitols were found to be active only toward α-galactosidase A. Finally, 1-N-iminosugars provided the best results, as 4-epi-isofagomine was found to be a good inhibitor of both lysosomal ß-galactosidase and GALC. Further elaboration of this structure is required to achieve selectivity between these two galactosidases.
Asunto(s)
Galactósidos/química , Galactosilceramidasa/antagonistas & inhibidores , Iminoazúcares/química , alfa-Galactosidasa/antagonistas & inhibidores , beta-Galactosidasa/antagonistas & inhibidores , Galactosilceramidasa/metabolismo , Humanos , Iminopiranosas/antagonistas & inhibidores , Iminopiranosas/metabolismo , Iminoazúcares/metabolismo , Iminoazúcares/uso terapéutico , Leucodistrofia de Células Globoides/tratamiento farmacológico , Lisosomas/enzimología , Unión Proteica , Relación Estructura-Actividad , alfa-Galactosidasa/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
The effect on the conformations and stability of gas-phase ions of Cerezyme, a glycoprotein, when bound to three small-molecule chaperones has been studied using intact ESI MS, collision cross section and MS/MS measurements. To distinguish between the peaks from apo and small-molecule complex ions, Cerezyme is deglycosylated (dg-Cer). ESI MS of dg-Cer reveals that glycosylation accounts for 8.5% of the molecular weight. When excess chaperone, either covalent (2FGF) or noncovalent (A and B iminosugars), is added to solutions of dg-Cer, mass spectra show peaks from 1:1 chaperone-enzyme complexes as well as free enzyme. On average, ions of the apoenzyme have 1.6 times higher cross sections when activated in the source region of the mass spectrometer. For a given charge state, ions of complexes of 2FGF and B have about 30% and 8.4% lower cross sections, respectively, compared to the apoenzyme. Thus, binding the chaperones causes the gas-phase protein to adopt more compact conformations. The noncovalent complex ions dissociate by the loss of charged chaperones. In the gas phase, the relative stability of dg-Cer with B is higher than that with the A, whereas in solution A binds enzyme more strongly than B. Nevertheless, the disagreement is explained based on the greater number of contacts between the B and dg-Cer than the A and dg-Cer (13 vs. 8), indicating the importance of noncovalent interactions within the protein-chaperone complex in the absence of solvent. Findings in this work suggest a hypothesis towards predicting a consistent correlation between gas-phase properties to solution binding properties.
Asunto(s)
Glucosilceramidasa/química , Iminoazúcares/química , Secuencia de Aminoácidos , Desoxiglucosa/análogos & derivados , Gases/química , Glucosilceramidasa/metabolismo , Glicosilación , Iminoazúcares/metabolismo , Iones/química , Modelos Moleculares , Datos de Secuencia Molecular , Espectrometría de Masas en TándemRESUMEN
A water extract of the leaves of Suregada glomerulata (Euphorbiaceae) was found to inhibit rat small intestinal α-glucosidase. An examination of the extract afforded 20 iminosugars including one pyrrolidine and 19 piperidines. The structures of the 10 new compounds (11-20) were determined by NMR, and MS spectroscopic data analyses, and chemical correlations. The novelty of the identified compounds mainly stems from the loss of a hydroxy at C-4 and the presence of an 8-hydroxyoctyl side chain. Nine N-alkyl derivatives including N-methyl (1a, 8a, and 13a), N-butyl (1b, 2b, and 9b) and N,N-dimethyl (1c, 2c, and 9c) were synthesized. The compounds were tested for rat small intestinal α-glucosidase inhibitory activity. In total, 15 compounds, including compounds 11, 12, 15, and 19 and the three derivatives 8a, 9b, and 13a, showed inhibitory activity with IC50 values less than 40µM. In vivo results showed that total alkaloids of S. glomerulata (10mg/kg) and four major iminosugars 1, 2, 3, and 9 (10mg/kg) can lower the postprandial blood glucose level after sucrose and starch load in healthy male ICR mice.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores de Glicósido Hidrolasas , Hipoglucemiantes/química , Iminoazúcares/química , Suregada/química , Animales , Glucemia/análisis , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/metabolismo , Hipoglucemiantes/uso terapéutico , Iminoazúcares/aislamiento & purificación , Iminoazúcares/metabolismo , Intestino Delgado/enzimología , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos ICR , Conformación Molecular , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Unión Proteica , Ratas , Suregada/metabolismo , alfa-Glucosidasas/metabolismoRESUMEN
In view of recent reports of a strong multivalent effect in glycosidase inhibition, a library of ß-CD-based multivalent iminosugars has been efficiently synthesized by way of Cu(I) -catalyzed azide-alkyne cycloaddition (CuAAC). In combination with the first application of isothermal titration calorimetry (ITC) experiments to the study of multivalent iminosugar-enzyme interactions, the inhibition properties of these click clusters were evaluated on a panel of glycosidases. The structural parameters that were varied include valency, peripheral ligand structure, and topology. The inhibition results obtained with the iminosugar clusters further highlight the importance of multivalency in the inhibition of α-mannosidase. Generally, the evaluated multivalent iminosugars displayed comparable thermodynamic signatures of binding towards α-mannosidase (Jack bean): that is, large negative enthalpies of complexation coupled with small entropies of either sign. In addition, the enthalpy-entropy compensation observed in all tested cases may be attributed to a common mechanism of dissociation for the enzyme-multivalent iminosugar interactions. The measured binding stoichiometries indicated that each iminosugar cluster interacts with no more than one protein molecule.
Asunto(s)
Química Clic , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/metabolismo , Iminoazúcares/química , Iminoazúcares/farmacología , beta-Ciclodextrinas/química , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Iminoazúcares/síntesis química , Iminoazúcares/metabolismo , Ligandos , Relación Estructura-Actividad , TermodinámicaRESUMEN
The rapid discovery of ß-glucocerebrosidase (GCase) inhibitors and pharmacological chaperones for Gaucher disease is described. The N-aminobutyl DNJ-based iminosugar was synthesized and conjugating with a variety of carboxylic acids to generate a N-diversely substituted iminosugar-based library. Several members of this library were found to be nanomolar-range inhibitors of GCase; the inhibition constant Ki of the most potent was found to be 71nM. Although these new molecules showed reasonable chaperoning activity (1.5- to 1.9-fold) in the N370S fibroblast of Gaucher patient-derived cell line, this was accompanies by a concomitant decrease in the cellular α-glucosidase activity, which might limit their further therapeutic potential. Next, newly developed N-substituents were assembled with pyrrolidine-based scaffolds to generate new molecules for further evaluation. The new 2,5-dideoxy-2,5-imino-d-mannitol (DMDP)-based iminosugar 22 was found to exhibit a satisfactory chaperoning activity to enhance GCase activity by 2.2-fold in Gaucher N370S cell line, without impairment of cellular α-glucosidase activity.
Asunto(s)
Inhibidores Enzimáticos/química , Enfermedad de Gaucher/enzimología , Glucosilceramidasa/antagonistas & inhibidores , Iminoazúcares/química , Sitios de Unión , Línea Celular , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Enfermedad de Gaucher/patología , Glucosilceramidasa/metabolismo , Humanos , Iminoazúcares/síntesis química , Iminoazúcares/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes with three (EH3I), two (EH2I), one (EHI), or no protons (EI), are possible. In the present work an analysis method is presented that from pH-inhibition data allows one to distinguish between the different complexes and determine which protonation state is preferred. It is also possible to determine the pH-independent binding constants of the inhibitor. Analysis of pH data for imino- and azasugar inhibition of ß-glucosidases revealed that basic glycosidase inhibitors bind as the monoprotonated (EHI) complex. Three neutral inhibitors were also studied and two of these were also bound exclusively as the EHI complex while a third bound both as a EHI and a EH2I complex.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Glicósido Hidrolasas/metabolismo , Iminoazúcares/metabolismo , Compuestos Aza/química , Dominio Catalítico , Inhibidores Enzimáticos/química , Glicósido Hidrolasas/antagonistas & inhibidores , Concentración de Iones de Hidrógeno , Iminoazúcares/química , Cinética , Unión Proteica , ProtonesRESUMEN
The effects of binding two small-molecule inhibitors to Agrobacterium sp. strain ATCC 21400 (Abg) ß-glucosidase on the conformations and stability of gas-phase ions of Abg have been investigated. Biotin-iminosugar conjugate (BIC) binds noncovalently to Abg while 2,4-dinitro-2-deoxy-2-fluoro-ß-D-glucopyranoside (2FG-DNP) binds covalently with loss of DNP. In solution, Abg is a dimer. Mass spectra show predominantly dimer ions, provided care is taken to avoid dissociation of dimers in solution and dimer ions in the ion sampling interface. When excess inhibitor, either covalent or noncovalent, is added to solutions of Abg, mass spectra show peaks almost entirely from 2:2 inhibitor-enzyme dimer complexes. Tandem mass spectrometry experiments show similar dissociation channels for the apo-enzyme and 2FG-enzyme dimers. The +21 dimer produces +10 and +11 monomers. The internal energy required to dissociate the +21 2FG-enzyme to its monomers (767 ± 30 eV) is about 36 eV higher than that for the apo-enzyme dimer (731 ± 6 eV), reflecting the stabilization of the free enzyme dimer by the 2FG inhibitor. The primary dissociation channels for the noncovalent BIC-enzyme dimer are loss of neutral and charged BIC. The internal energy required to induce loss of BIC is 482 ± 8 eV, considerably less than that required to dissociate the dimers. For a given charge state, ions of the covalent and noncovalent complexes have about 15 % and 25 % lower cross sections, respectively, compared with the apo-enzyme. Thus, binding the inhibitors causes the gas-phase protein to adopt more compact conformations. Noncovalent binding surprisingly produces the greatest change in protein ion conformation, despite the weaker inhibitor binding. á
Asunto(s)
Espectrometría de Masas en Tándem/métodos , beta-Glucosidasa/antagonistas & inhibidores , beta-Glucosidasa/química , Agrobacterium/enzimología , Biotina/química , Biotina/metabolismo , Gases/química , Glucósidos/química , Glucósidos/metabolismo , Iminoazúcares/química , Iminoazúcares/metabolismo , Iones/química , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , beta-Glucosidasa/metabolismoRESUMEN
The mirror image of natural product (+)-adenophorine along with its 1-epi-, 1-homo-analogs and other derivatives have been synthesized and evaluated as glycosidase inhibitors. The synthetic strategy is based on the skeletal rearrangement of tetrahydroxylated C-alkyl azepanes obtained via a Staudinger/azaWittig/alkylation sequence starting from a sugar-derived azidolactol. Several organometallic species have been investigated for the alkylation step including organomagnesium, organolithium, organozinc, organoaluminum and organocerium reagents. While diallylzinc proved to be the most efficient to introduce an allyl substituent, disappointing results were obtained with the other organometallic species leading either to lower yields or no reaction. Enzymatic assays indicate that (-)-adenophorine is a moderate α-l-fucosidase inhibitor.
Asunto(s)
Compuestos Aza/síntesis química , Inhibidores Enzimáticos/síntesis química , Glicósido Hidrolasas/antagonistas & inhibidores , Iminoazúcares/química , Alquilación , Animales , Aspergillus niger/enzimología , Compuestos Aza/química , Compuestos Aza/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Glicósido Hidrolasas/metabolismo , Iminoazúcares/síntesis química , Iminoazúcares/metabolismo , Oryza/enzimología , Unión Proteica , Ratas , Saccharomyces cerevisiae/enzimología , Estereoisomerismo , alfa-L-Fucosidasa/antagonistas & inhibidores , alfa-L-Fucosidasa/metabolismoRESUMEN
Forty years of discoveries and research on imino sugars, which are carbohydrate analogues having a basic nitrogen atom instead of oxygen in the sugar ring and, acting as potent glycosidase inhibitors, have made considerable impact on our contemporary understanding of glycosidases. Imino sugars have helped to elucidate the catalytic machinery of glycosidases and have refined our methods and concepts of utilizing them. A number of new aspects have emerged for employing imino sugars as pharmaceutical compounds, based on their profound effects on metabolic activities in which glycosidases are involved. From the digestion of starch to the fight against viral infections, from research into malignant diseases to potential improvements in hereditary storage disorders, glycosidase action and inhibition are essential issues. This account aims at combining general developments with a focus on some niches where imino sugars have become useful tools for glycochemistry and glycobiology.
Asunto(s)
Inhibidores Enzimáticos , Glicósido Hidrolasas , Iminoazúcares , Animales , Biocatálisis , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Humanos , Iminoazúcares/química , Iminoazúcares/metabolismo , Iminoazúcares/farmacologíaRESUMEN
Recently, pharmacological chaperone therapy for Pompe disease with small molecules such as imino sugars has attracted interest. But mutant acid α-glucosidase (GAA) species responsive to imino sugars are limited. To elucidate the characteristics of a mutant GAA responsive to imino sugars, we performed biochemical and structural analyses. Among cultured fibroblast cell lines derived from Japanese Pompe patients, only one carrying p.S529V/p.S619R amino acid substitutions responded to 1-deoxynojirimycin (DNJ), and an expression study revealed that DNJ, N-butyl-deoxynojirimycin and nojirimycin-1-sulfonic acid increased the enzyme activity of the S529V mutant GAA expressed in Chinese hamster ovary cells. The results of western blotting analysis suggested that these imino sugars facilitated the intracellular transportation of the mutant GAA and stabilized it. Among these imino sugars, DNJ exhibited the strongest action on the mutant GAA. Structural analysis revealed that DNJ almost completely occupied the active site pocket, and interacted with amino acid residues comprising it through van der Waals contacts and hydrogen bonds. This information will be useful for improvement of pharmacological chaperone therapy for Pompe disease.
Asunto(s)
Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , alfa-Glucosidasas/química , alfa-Glucosidasas/metabolismo , Sustitución de Aminoácidos/genética , Animales , Células CHO , Dominio Catalítico/genética , Células Cultivadas , Cricetinae , Cricetulus , Activación Enzimática/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Humanos , Iminoazúcares/metabolismo , Iminoazúcares/farmacología , Modelos Moleculares , Unión Proteica , Conformación Proteica , alfa-Glucosidasas/genéticaRESUMEN
Small molecules including imino sugars are expected to act as chaperones for a mutant α-galactosidase A (GLA), which will be useful for pharmacological chaperone therapy for Fabry disease. However, there is little detailed information about the molecular mechanism. We paid attention to an M51I mutant GLA which had been reported to strongly react to an imino sugar. The predicted structural change caused by this amino acid substitution is very small and located on the surface of the molecule. We produced the mutant enzyme in yeast, and determined its enzymological characteristics. The enzymological parameter values are almost the same as those of the wild-type GLA, although the mutant enzyme is unstable not only under neutral pH conditions but also under acidic ones. Then, we directly examined the effect of imino sugars including 1-deoxygalactonojirimycin and galactostatin bisulfite on the purified mutant enzyme. The imino sugars apparently improved the stability of the mutant enzyme under both neutral and acidic pH conditions. The results of surface plasmon resonance biosensor assaying suggested that the imino sugars retained their binding activity as to the mutant enzyme under both neutral and acidic pH conditions. This information will facilitate improvement of pharmacological chaperone therapy for Fabry disease.