RESUMO
A new class of compounds, namely highly substituted diaminocyclopentane-l-lysine adducts, have been discovered as potent inhibitors of O-GlcNAcase, an enzyme crucial for protein de-O-glycosylation. These inhibitors exhibit exceptional selectivity and reversibility and are the first example of human O-GlcNAcase inhibitors that are structurally related to the transition state of the rate-limiting step with the "aglycon" still in bond-length proximity. The ease of their preparation, remarkable biological activities, stability, and non-toxicity make them promising candidates for the development of anti-tau-phosphorylation agents holding significant potential for the treatment of Alzheimer's disease.
Assuntos
Inibidores Enzimáticos , Lisina , Humanos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Relação Estrutura-Atividade , Lisina/química , Lisina/farmacologia , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismo , Ciclopentanos/química , Ciclopentanos/farmacologia , Ciclopentanos/síntese química , Estrutura Molecular , Relação Dose-Resposta a DrogaRESUMO
Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity-based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity. Here, we report in a proof of concept study the use of ligand-directed chemistry (LDC) for labelling glycoside hydrolases near - but not in - the active site. During the labelling process, the competitive inhibitor is cleaved from the probe, departs the active site and the enzyme maintains its catalytic activity. To this end, we designed a building block synthetic concept for small molecule probes containing iminosugar-based reversible inhibitors for labelling of two model ß-glucosidases. The results indicate that the LDC approach can be adaptable for covalent proximity labelling of glycoside hydrolases.
Assuntos
Carboidratos , Glicosídeo Hidrolases , Glicosídeo Hidrolases/metabolismo , Estudo de Prova de Conceito , LigantesRESUMO
A new class of compounds inhibiting de-O-glycosylation of proteins has been identified. Highly substituted diaminocyclopentanes are impressively selective reversible non-transition state O-ß-N-acetyl-d-glucosaminidase (O-GlcNAcase) inhibitors. The ease of preparative access and remarkable biological activities provide highly viable leads for the development of anti-tau-phosphorylation agents with a view to eventually ameliorating Alzheimer's disease.
Assuntos
Doença de Alzheimer , beta-N-Acetil-Hexosaminidases , Humanos , Hexosaminidases , GlicosilaçãoRESUMO
A short survey on selected ß-galactosidase inhibitors as potential pharmacological chaperones for GM1 -gangliosidosis and Morquio B associated mutants of human lysosomal ß-galactosidase is provided highlighting recent developments in this particular area of lysosomal storage disorders and orphan diseases.
Assuntos
Gangliosidoses , Gangliosidose GM1 , Mucopolissacaridose IV , beta-Galactosidase/antagonistas & inibidores , Gangliosidose GM1/tratamento farmacológico , Humanos , Lisossomos , Mucopolissacaridose IV/tratamento farmacológicoRESUMO
The scope of a series of N-alkylated iminosugar based inhibitors in the d-gluco as well as d-xylo configuration towards their interaction with human lysosomal ß-glucocerebrosidase has been evaluated. A versatile synthetic toolbox has been developed for the synthesis of N-alkylated iminosugar scaffolds conjugated to a variety of terminal groups via a benzoic acid ester linker. The terminal groups such as nitrile, azide, alkyne, nonafluoro-tert-butyl and amino substituents enable follow-up chemistry as well as visualisation experiments. All compounds showed promising inhibitory properties as well as selectivities for ß-glucosidases, some exhibiting activities in the low nanomolar range for ß-glucocerebrosidase.
Assuntos
Glucosilceramidase/metabolismo , Lisossomos/enzimologia , Ácido Benzoico/metabolismo , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid ß-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent ß-d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a "strategic" hydroxyl group. New compounds have revealed highly promising activities with a range of ß-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.
Assuntos
Ciclopentanos/farmacologia , Galactosidases/metabolismo , Imino Piranoses/farmacologia , Lisossomos/enzimologia , Chaperonas Moleculares/metabolismo , Cristalização , Ciclopentanos/síntese química , Ciclopentanos/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Galactosidases/antagonistas & inibidores , Humanos , Imino Piranoses/síntese química , Imino Piranoses/química , Ligantes , Lisossomos/efeitos dos fármacos , Conformação Molecular , Proteínas Mutantes/metabolismoRESUMO
N-Functionalized amino(hydroxymethyl)cyclopentanetriols are potent inhibitors of ß-d-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid ß-galactosidase thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.
Assuntos
Ciclopentanos/química , Ciclopentanos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Gangliosidose GM1/tratamento farmacológico , beta-Galactosidase/antagonistas & inibidores , Aminação , Animais , Bovinos , Gangliosidose GM1/enzimologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Metilação , beta-Galactosidase/metabolismoRESUMO
From an easily available partially protected formal derivative of 1-deoxymannojirimycin, by hydroxymethyl chain-branching and further elaboration, lipophilic analogs of the powerful ß-d-galactosidase inhibitor 4-epi-isofagomine have become available. New compounds exhibit improved inhibitory activities comparable to benchmark compound NOEV (N-octyl-epi-valienamine) and may serve as leads towards improved and more selective pharmacological chaperones for GM1-gangliosidosis.
Assuntos
Inibidores Enzimáticos/farmacologia , Gangliosidose GM1/enzimologia , Imino Piranoses/farmacologia , Lisossomos/enzimologia , beta-Galactosidase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Gangliosidose GM1/patologia , Humanos , Imino Piranoses/síntese química , Imino Piranoses/química , Lisossomos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , beta-Galactosidase/metabolismoRESUMO
Isoiminosugars are highly biological active substances. Herein, we report a concise synthetic approach for this class of compounds. The key step relies on a stereospecific 1,2-hydride shift in O-2 tosylated glycopyranosides leading to C-2 branched glycofuranosides. This approach enables a 4-step synthesis of powerful ß-galactosidase inhibitor 4-epi-isofagomine starting from a simple d-glucopyranoside.
RESUMO
Yarrowia lipolytica short chain dehydrogenase/reductase (YlSDR) was expressed in Escherichia coli, purified and characterized in vitro. The substrate scope for YlSDR mediated oxidation was investigated with alcohols and unprotected carbohydrates spectrophotometrically, revealing a preference for secondary compared to primary alcohols. In reduction direction, YlSDR was highly active on ribulose and fructose, suggesting that the enzyme is a mannitol-2-dehydrogenase. In order to explore substrate tolerance especially for space-demanding, lipophilic protecting groups, 5-O-trityl-D-ribitol and 5-O-trityl-α,ß-D-ribose were investigated as substrates: YlSDR oxidized 5-O-trityl-D-ribitol and 5-O-trityl-α,ß-D-ribose and reduced the latter at the expense of NADP(H).
Assuntos
Ácido Graxo Sintases/metabolismo , NADH NADPH Oxirredutases/metabolismo , Pentoses/metabolismo , Ribitol/metabolismo , Yarrowia/enzimologia , Escherichia coli/metabolismo , Ácido Graxo Sintases/genética , Frutose/metabolismo , Cinética , Manitol Desidrogenases/genética , Manitol Desidrogenases/metabolismo , NADH NADPH Oxirredutases/genética , NADP/metabolismo , Oxirredução , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Ribose/metabolismo , Especificidade por SubstratoRESUMO
A set of cyclopentanoid α-galactosidase ligands was prepared from a partially protected ω-eno-aldose via a reliable (2 + 3)-cycloaddition protocol with slightly modified conditions. The obtained N-benzylisoxazolidine ring was selectively opened and the configuration of the hydroxymethylgroup was inverted. Consecutive deprotection provided an aminocyclopentane, which was N-alkylated to furnish a set of potential α-galactosidase inhibitors. Their glycosidase inhibitory activities were screened with a panel of standard glycosidases of biological significance.
RESUMO
We report here a one-step aqueous method for the synthesis of isolated and purified polysaccharide-amino acid conjugates. Two different types of amino acid esters: glycine methyl ester and L-tryptophan methyl ester, as model compounds for peptides, were conjugated to the polysaccharide carboxymethylcellulose (CMC) in water using carbodiimide at ambient conditions. Detailed and systematic pH-dependent charge titration and spectroscopy (infrared, nuclear magnetic resonance: 1H, 13C- DEPT 135, 1H- 13C HMBC/HSQC correlation), UV-vis, elemental and ninhydrin analysis provided solid and direct evidence for the successful conjugation of the amino acid esters to the CMC backbone via an amide bond. As the concentration of amino acid esters increased, a conjugation efficiency of 20-80% was achieved. Activated charcoal aided base-catalyzed deprotection of the methyl esters improved the solubility of the conjugates in water. The approach proposed in this work should have the potential to tailor the backbone of polysaccharides containing di- or tri-peptides.
Assuntos
Carbodi-Imidas/química , Carboximetilcelulose Sódica/análogos & derivados , Glicina/análogos & derivados , Indicadores e Reagentes/química , Triptofano/análogos & derivados , Carboximetilcelulose Sódica/síntese química , Glicina/síntese química , Estrutura Molecular , Triptofano/síntese químicaRESUMO
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.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Galactosidases/antagonistas & inibidores , Gangliosidose GM1/tratamento farmacológico , Álcoois Açúcares/química , Álcoois Açúcares/farmacologia , Linhagem Celular , Café/enzimologia , Inibidores Enzimáticos/uso terapêutico , Escherichia coli/enzimologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Galactosidases/metabolismo , Halogenação , Humanos , Iminas/química , Iminas/farmacologia , Iminas/uso terapêutico , Rhizobium/enzimologia , Álcoois Açúcares/uso terapêuticoRESUMO
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.
RESUMO
This work describes the derivatization of dextran using N-(tert-butyloxycarbonyl)-S-(trityl)-L-cysteine in the presence of N,N'-carbonyldiimidazole (CDI) as a coupling agent. Homogeneous reactions in dimethyl sulfoxide allowed for an efficient coupling of the amino acid derivative to the polymer backbone. Derivatization was confirmed by infrared and 13C NMR spectroscopy, size exclusion chromatography and elemental analysis. The presence of hydrophobic protecting groups resulted in a product that can be shaped into water-insoluble particles stable in an aqueous environment and non-toxic for lung epithelial cells. It is suggested that materials composed of ester bonds between amino acids and polysaccharides are useful for targeted drug delivery, bio-imaging or surface functionalization.
Assuntos
Aminoácidos/química , Cisteína/análogos & derivados , Ésteres/química , Cisteína/química , Dextranos/química , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
A series of N-alkyl derivatives of the D-galactosidase inhibitor 1,4-di-epi-validamine featuring lipophilic substituents at position C-5a was prepared and screened for their glycosidase inhibitory properties. Products turned out selective for ß-galactosidases as well as ß-glucosidases.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteínas Fúngicas/antagonistas & inibidores , Inositol/análogos & derivados , beta-Galactosidase/antagonistas & inibidores , beta-Glucosidase/antagonistas & inibidores , Agrobacterium/química , Agrobacterium/enzimologia , Animais , Proteínas de Bactérias/química , Configuração de Carboidratos , Bovinos , Inibidores Enzimáticos/síntese química , Escherichia coli/química , Escherichia coli/enzimologia , Proteínas Fúngicas/química , Interações Hidrofóbicas e Hidrofílicas , Inositol/síntese química , Inositol/química , Cinética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , beta-Galactosidase/química , beta-Glucosidase/químicaRESUMO
From 1,2;3,4-di-O-isopropylidene-α-D-galactopyranose, a series of highly functionalized (hydroxymethyl)cyclopentanes was easily available. In line with reports by Reymond and Jäger on similar structures, these amine containing basic carbasugars are potent inhibitors of ß-D-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for GM1-gangliosidosis-associated lysosomal acid ß-galactosidase mutant R201C, thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease.
Assuntos
Ciclopentanos/química , Ciclopentanos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Gangliosidose GM1/genética , Mutação , beta-Galactosidase/antagonistas & inibidores , Ciclopentanos/síntese química , Inibidores Enzimáticos/síntese química , Gangliosidose GM1/enzimologia , Humanos , Modelos Moleculares , Conformação Molecular , beta-Galactosidase/genéticaRESUMO
By Morita-Baylis-Hillman reaction of 2,3-O-isopropylidene-D-glyceraldehyde with α,ß-unsaturated carbonyl as well as hetero analogous carbonyl compounds such as acrylonitrile, suitable precursors of isofagomine and of 4-epi-isofagomine are available. Elaboration of the structures by amine introduction, followed by intramolecular ring closure and subsequent hydroboration of the double bond provides 4-epi-isofagomine derivatives featuring chain extensions at C-5a which are determined by the structures of the carbonyl compounds employed. As an example, the synthesis of C-(5aR)- and C-(5aS)-5a-C-pentyl-4-epi-isofagomines, powerful inhibitors of ß-galactosidases, is outlined. In line with reported data, the (C-5aR) epimer was found a highly potent experimental pharmacological chaperone for GM1-associated human lysosomal ß-galactosidase mutant R201C.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Imino Piranoses/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/metabolismo , Humanos , Imino Piranoses/síntese química , Imino Piranoses/química , Lisossomos/enzimologia , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
From a secondary hydroxyl group, by the simple sequence of oxidation, Wittig reaction of the obtained ulose with methoxymethylene triphenyl phosphorane, exposure of the resulting exocyclic enol ether to Selectfluor and subsequent reduction of the α-fluoro aldehyde thus obtained, tertiary fluoro substituents can be introduced into carbohydrate and carbohydrate-related scaffolds at a branching point now bearing a new hydroxymethyl group.
Assuntos
Álcoois/química , Carboidratos/química , Fluoretos/química , Flúor/química , Estrutura Molecular , Oxirredução , EstereoisomerismoRESUMO
Electrophilic fluorination of an exocyclic methoxymethylene enol ether derived from N-tert-butyloxycarbonyl-1,5-dideoxy-1,5-imino-3,4-O-isopropylidene-D-erythro-pent-2-ulose (11) provided the 5-fluoro derivative of the powerful ß-galactosidase inhibitor 4-epi-isofagomine (8). This structural alteration, in combination with N-alkylation, led to considerably improved α-galactosidase selectivity. New compounds may serve as leads en route to new pharmacological chaperones for Fabry's disease.