RESUMO
A remarkable enhancer of human glucocerebrosidase enzyme (GCase) was identified among a set of dihydroazulene-tagged iminosugars. An unprecedented 3.9-fold increase in GCase activity was detected on fibroblasts bearing the homozygous L444P mutation, which is frequently associated with neuronopathic Gaucher forms, and which commonly results refractory to chaperone-induced refolding.
Assuntos
Glucosilceramidase , Mutação , Humanos , Glucosilceramidase/metabolismo , Glucosilceramidase/genética , Glucosilceramidase/antagonistas & inibidores , Imino Açúcares/química , Imino Açúcares/farmacologia , Imino Açúcares/síntese química , Imino Açúcares/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Doença de Gaucher/genética , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/metabolismo , Estrutura MolecularRESUMO
Gaucher disease (GD) is a rare genetic metabolic disorder characterized by a dysfunction of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) due to mutations in the gene GBA1, leading to the cellular accumulation of glucosylceramide (GlcCer). While most of the current research focuses on the primary accumulated material, lesser attention has been paid to secondary storage materials and their reciprocal intertwining. By using a novel approach based on flow cytometry and fluorescent labelling, we monitored changes in storage materials directly in fibroblasts derived from GD patients carrying N370S/RecNcil and homozygous L444P or R131C mutations with respect to wild type. In L444P and R131C fibroblasts, we detected not only the primary accumulation of GlcCer accumulation but also a considerable secondary increase in GM1 storage, comparable with the one observed in infantile patients affected by GM1 gangliosidosis. In addition, the ability of a trivalent trihydroxypiperidine iminosugar compound (CV82), which previously showed good pharmacological chaperone activity on GCase enzyme, to reduce the levels of storage materials in L444P and R131C fibroblasts was tested. Interestingly, treatment with different concentrations of CV82 led to a significant reduction in GM1 accumulation only in L444P fibroblasts, without significantly affecting GlcCer levels. The compound CV82 was selective against the GCase enzyme with respect to the ß-Galactosidase enzyme, which was responsible for the catabolism of GM1 ganglioside. The reduction in GM1-ganglioside level cannot be therefore ascribed to a direct action of CV82 on ß-Galactosidase enzyme, suggesting that GM1 decrease is rather related to other unknown mechanisms that follow the direct action of CV82 on GCase. In conclusion, this work indicates that the tracking of secondary storages can represent a key step for a better understanding of the pathways involved in the severity of GD, also underlying the importance of developing drugs able to reduce both primary and secondary storage-material accumulations in GD.
Assuntos
Gangliosídeo G(M1) , Doença de Gaucher , Humanos , Fibroblastos , beta-Galactosidase/genética , Corantes , Citometria de Fluxo , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , GlucosilceramidasRESUMO
Engineering bioactive iminosugars with pH-responsive groups is an emerging approach to develop pharmacological chaperones (PCs) able to improve lysosomal trafficking and enzymatic activity rescue of mutated enzymes. The use of inexpensive l-malic acid allowed introduction of orthoester units into the lipophilic chain of an enantiomerically pure iminosugar affording only two diastereoisomers contrary to previous related studies. The iminosugar was prepared stereoselectively from the chiral pool (d-mannose) and chosen as the lead bioactive compound, to develop novel candidates for restoring the lysosomal enzyme glucocerebrosidase (GCase) activity. The stability of orthoester-appended iminosugars was studied by 1 Hâ NMR spectroscopy both in neutral and acidic environments, and the loss of inhibitory activity with time in acid medium was demonstrated on cell lysates. Moreover, the ability to rescue GCase activity in the lysosomes as the result of a chaperoning effect was explored. A remarkable pharmacological chaperone activity was measured in fibroblasts hosting the homozygous L444P/L444P mutation, a cell line resistant to most PCs, besides the more commonly responding N370S mutation.
Assuntos
Doença de Gaucher , Glucosilceramidase , Humanos , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Piperidinas/farmacologia , Piperidinas/metabolismo , Mutação , Fibroblastos , Concentração de Íons de HidrogênioRESUMO
N-Acetylgalactosamine-6-sulfatase (GALNS) is an enzyme whose deficiency is related to the lysosomal storage disease Morquio A. For the development of effective therapeutic approaches against this disease, the design of suitable enzyme enhancers (i.e. pharmacological chaperones) is fundamental. The natural substrates of GALNS are the glycosaminoglycans keratan sulfate and chondroitin 6-sulfate, which mainly display repeating units of sulfated carbohydrates. With a biomimetic approach, gold nanoparticles (AuNPs) decorated with simple monosaccharides, sulfated ligands (homoligand AuNPs), or both monosaccharides and sulfated ligands (mixed-ligand AuNPs) were designed here as multivalent inhibitors of GALNS. Among the homoligand AuNPs, the most effective inhibitors of GALNS activity are the ß-D-galactoside-coated AuNPs. In the case of mixed-ligand AuNPs, ß-D-galactosides/sulfated ligands do not show better inhibition than the ß-D-galactoside-coated AuNPs. However, a synergistic effect is observed for α-D-mannosides in a mixed-ligand coating with sulfated ligands that reduced IC50 by one order of magnitude with respect to the homoligand α-D-mannoside-coated AuNPs. SAXS experiments corroborated the association of GALNS with ß-D-galactoside AuNPs. These AuNPs are able to restore the enzyme activity by almost 2-fold after thermal denaturation, indicating a potential chaperoning activity towards GALNS. This information could be exploited for future development of nanomedicines for Morquio A. The recent implications of GALNS in cancer and neuropathic pain make these kinds of multivalent bionanomaterials of great interest towards multiple therapies.
Assuntos
Condroitina Sulfatases , Nanopartículas Metálicas , Ouro , Acetilgalactosamina , Monossacarídeos , Ligantes , Sulfatos , Espalhamento a Baixo Ângulo , Difração de Raios X , LisossomosRESUMO
A novel stereoselective synthetic approach to pentahydroxyazepane iminosugars is described. The strategy relies on a key osmium-catalyzed aminohydroxylation reaction of allylic alcohols obtained via addition of vinylmagnesium bromide to a d-mannose-derived aldehyde, which forms the new C-N bond with complete regio- and stereocontrol according to the tethering approach. Subsequent intramolecular reductive amination afforded the desired azepanes. This method represents the first application of the osmium-catalyzed tethered aminohydroxylation reaction to the synthesis of iminosugars.
RESUMO
A collection of novel mono- and three-tailed derivatives based on a sugar (glucose) or an iminosugar (trihydroxy piperidine) featuring a terminal benzenesulfonamide were synthesized to investigate the so-called "sugar" and "azasugar" approach with the aim of exploring the activity and selectivity towards the inhibition of human carbonic anhydrases (hCAs). The synthetic approach relies on a general copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction followed by an amine-isothiocyanate coupling. Biological assays were used to collect subtle information on the role of these single or multiple hydrophilic chains. Among the sugar-based inhibitors, the single-tailed compound 10 was identified as a better inhibitor than the reference compound (AAZ) towards three different hCAs, while, among the three sugar tailed derivatives, potent and selective inhibition was found for compounds 25 and 26. A promising and selective inhibitory activity was discovered for the iminosugar single-tailed compound 31 towards hCA VII (Ki = 9.7 nM).
Assuntos
Inibidores da Anidrase Carbônica , Anidrases Carbônicas , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Inibidores da Anidrase Carbônica/farmacologia , Açúcares , Relação Dose-Resposta a Droga , Anidrases Carbônicas/metabolismo , Anidrase Carbônica IX/metabolismo , BenzenossulfonamidasRESUMO
Piperidine-based photoswitchable derivatives have been developed as putative pharmacological chaperones for glucocerebrosidase (GCase), the defective enzyme in Gaucher disease (GD). The structure-activity study revealed that both the iminosugar and the light-sensitive azobenzene are essential features to exert inhibitory activity towards human GCase and a system with the correct inhibition trend (IC50 of the light-activated form lower than IC50 of the dark form) was identified. Kinetic analyses showed that all compounds are non-competitive inhibitors (mixed or pure) of GCase and the enzyme allosteric site involved in the interaction was identified by means of MD simulations. A moderate activity enhancement of mutant GCase assessed in GD patients' fibroblasts (ex vivo experiments) carrying the most common mutation was recorded. This promising observation paves the way for further studies to improve the benefit of the light-to-dark thermal conversion for chaperoning activity.
Assuntos
Doença de Gaucher , Glucosilceramidase , Humanos , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Dobramento de Proteína , Fibroblastos/metabolismo , Mutação , Inibidores Enzimáticos/farmacologiaRESUMO
A general method for the synthesis of pyrrolizidine derivatives using an intramolecular hydroaminomethylation protocol (HAM) under microwave (MW) dielectric heating is reported. Starting from a 3,4-bis(benzyloxy)-2-[(benzyloxy)methyl]-5-vinylpyrrolidine, MW-assisted intramolecular HAM in the presence of gaseous H2 and CO gave the natural alkaloid hyacinthacine A2 protected as benzyl ether. The same approach gave a lentiginosine analogue starting from the corresponding vinyl N-hydroxypyrrolidine. The nature of the reaction products and the yields were strongly influenced by the relative stereochemistry of the starting pyrrolidines, as well as by the catalyst/ligand employed. The use of ethanol as a solvent provides environmentally friendly conditions, while the ligand/catalyst system can be recovered by separating the alkaloid product with an SCX column and recycling the ethanolic solution. HAM worked up to three times with the recycled catalyst solution without any significant impact on yield.
Assuntos
Alcaloides , Alcaloides de Pirrolizidina , Alcaloides/química , Calefação , Ligantes , Micro-Ondas , Alcaloides de Pirrolizidina/químicaRESUMO
Pharmaceutical chaperones (PCs) are small compounds able to bind and stabilize misfolded proteins, allowing them to recover their native folding and thus their biological activity. In particular, lysosomal storage disorders (LSDs), a class of metabolic disorders due to genetic mutations that result in misfolded lysosomal enzymes, can strongly benefit from the use of PCs able to facilitate their translocation to the lysosomes. This results in a recovery of their catalytic activity. No PC for the GCase enzyme (lysosomal acid-ß-glucosidase, or glucocerebrosidase) has reached the market yet, despite the importance of this enzyme not only for Gaucher disease, the most common LSD, but also for neurological disorders, such as Parkinson's disease. This review aims to describe the efforts made by the scientific community in the last 7 years (since 2015) in order to identify new PCs for the GCase enzyme, which have been mainly identified among glycomimetic-based compounds.
RESUMO
GM1 gangliosidosis is a rare lysosomal disease caused by the deficiency of the enzyme ß-galactosidase (ß-Gal; GLB1; E.C. 3.2.1.23), responsible for the hydrolysis of terminal ß-galactosyl residues from GM1 ganglioside, glycoproteins, and glycosaminoglycans, such as keratan-sulfate. With the aim of identifying new pharmacological chaperones for GM1 gangliosidosis, the synthesis of five new trihydroxypiperidine iminosugars is reported in this work. The target compounds feature a pentyl alkyl chain in different positions of the piperidine ring and different absolute configurations of the alkyl chain at C-2 and the hydroxy group at C-3. The organometallic addition of a Grignard reagent onto a carbohydrate-derived nitrone in the presence or absence of a suitable Lewis Acid was exploited, providing structural diversity at C-2, followed by the ring-closure reductive amination step. An oxidation-reduction process allowed access to a different configuration at C-3. The N-pentyl trihydroxypiperidine iminosugar was also synthesized for the purpose of comparison. The biological evaluation of the newly synthesized compounds was performed on leucocyte extracts from healthy donors and identified two suitable ß-Gal inhibitors, namely compounds 10 and 12. Among these, compound 12 showed chaperoning properties since it enhanced ß-Gal activity by 40% when tested on GM1 patients bearing the p.Ile51Asn/p.Arg201His mutations.
Assuntos
Gangliosidose GM1 , Gangliosidose GM1/tratamento farmacológico , Gangliosidose GM1/genética , Humanos , Lisossomos , Chaperonas Moleculares/genética , Mutação , beta-Galactosidase/químicaRESUMO
We are very happy to present this Special Issue, for which we acted as guest editors, and which includes scientific contributions from laboratories headed by women active in the field of bioorganic chemistry [...].
Assuntos
Laboratórios , Feminino , HumanosRESUMO
The synthesis of five new multivalent derivatives of a trihydroxypiperidine iminosugar was accomplished through copper catalyzed alkyne-azide cycloaddition (CuAAC) reaction of an azido ending piperidine and several propargylated scaffolds. The resulting multivalent architectures were assayed as inhibitors of lysosomal GCase, the defective enzyme in Gaucher disease. The multivalent compounds resulted in much more potent inhibitors than a parent monovalent reference compound, thus showing a good multivalent effect. Biological investigation of these compounds as pharmacological chaperones revealed that the trivalent derivative (12) gives a 2-fold recovery of the GCase activity on Gaucher patient fibroblasts bearing the L444P/L444P mutations responsible for neuropathies. Additionally, a thermal denaturation experiment showed its ability to impart stability to the recombinant enzyme used in therapy.
Assuntos
Doença de Gaucher , Glucosilceramidase , Inibidores Enzimáticos/farmacologia , Fibroblastos , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Glucosilceramidase/genética , Glucosilceramidase/uso terapêutico , Humanos , MutaçãoRESUMO
Light-switchable inhibitors of the enzyme ß-glucocerebrosidase (GCase) have been developed by anchoring a specific azasugar to a dihydroazulene or an azobenzene responsive moiety. Their inhibitory effect towards human GCase, before and after irradiation are reported, and the effect on thermal denaturation of recombinant GCase and cytotoxicity were studied on selected candidates.
Assuntos
Compostos Azo/farmacologia , Azulenos/farmacologia , Inibidores Enzimáticos/farmacologia , Glucosilceramidase/antagonistas & inibidores , Compostos Azo/síntese química , Compostos Azo/química , Azulenos/síntese química , Azulenos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glucosilceramidase/metabolismo , Humanos , Luz , Estrutura Molecular , Processos FotoquímicosRESUMO
Among carbohydrate-processing enzymes, Jack bean α-mannosidase (JBα-man) is the glycosidase with the best responsiveness to the multivalent presentation of iminosugar inhitopes. We report, in this work, the preparation of water dispersible gold nanoparticles simultaneously coated with the iminosugar deoxynojirimycin (DNJ) inhitope and simple monosaccharides (ß-d-gluco- or α-d-mannosides). The display of DNJ at the gold surface has been modulated (i) by using an amphiphilic linker longer than the aliphatic chain used for the monosaccharides and (ii) by presenting the inhitope, not only in monomeric form, but also in a trimeric fashion through combination of a dendron approach with glyconanotechnology. The latter strategy resulted in a strong enhancement of the inhibitory activity towards JBα-man, with a Ki in the nanomolar range (Ki = 84 nM), i.e., more than three orders of magnitude higher than the monovalent reference compound.
Assuntos
1-Desoxinojirimicina/administração & dosagem , Canavalia/enzimologia , Inibidores Enzimáticos/administração & dosagem , Ouro/química , Nanopartículas Metálicas/administração & dosagem , alfa-Manosidase/antagonistas & inibidores , 1-Desoxinojirimicina/química , Inibidores Enzimáticos/química , Nanopartículas Metálicas/químicaRESUMO
We report a straightforward synthetic strategy for the preparation of trihydroxypiperidine azasugars decorated with lipophilic chains at both the nitrogen and the adjacent carbon as potential inhibitors of the lysosomal enzyme glucocerebrosidase (GCase), which is involved in Gaucher disease. The procedure relies on the preparation of C-erythrosyl N-alkylated nitrones 10 through reaction of aldehyde 8 and primary amines 13 followed by oxidation of the imines formed in situ with the methyltrioxorhenium catalyst and urea hydrogen peroxide. The addition of octylMgBr to nitrone 10e provided access to both epimeric hydroxylamines 21 and 22 with opposite configuration at the newly created stereocenter in a stereodivergent and completely stereoselective way, depending on the absence or presence of BF3·Et2O. Final reductive amination and acetonide deprotection provided compounds 14 and 15 from low-cost d-mannose in remarkable 43 and 32% overall yields, respectively, over eight steps. The C-2 R-configured bis-alkylated trihydroxypiperidine 15 was the best ligand for GCase (IC50 = 15 µM), in agreement with MD simulations that allowed us to identify the chair conformation corresponding to the best binding affinity.
Assuntos
Doença de Gaucher , Glucosilceramidase , Aminação , Doença de Gaucher/tratamento farmacológico , Humanos , Oxirredução , PiperidinasRESUMO
The [3,3]-sigmatropic allyl cyanate/isocyanate rearrangement of glycals in the presence of O-, N-, and C-nucleophiles afforded ß-N-glucosyl and galactosyl carbamates, ureas, and amides in good yields. The unsaturated products were elaborated to N-glycosides by dihydroxylation, to 1,3-diaminosugars by tethered aminohydroxylation, or to 1,2-diaminosugars by iteration of the sigmatropic rearrangement. This metal-free methodology represents an excellent and general method for the stereoselective synthesis of N-glycosides and diamino sugars with complete transmission of stereochemical information.
RESUMO
Pharmacological chaperones (PCs) are small compounds able to rescue the activity of mutated lysosomal enzymes when used at subinhibitory concentrations. Nitrogen-containing glycomimetics such as aza- or iminosugars are known to behave as PCs for lysosomal storage disorders (LSDs). As part of our research into lysosomal sphingolipidoses inhibitors and looking in particular for new ß-galactosidase inhibitors, we report the synthesis of a series of alkylated azasugars with a relative "all-cis" configuration at the hydroxy/amine-substituted stereocenters. The novel compounds were synthesized from a common carbohydrate-derived piperidinone intermediate 8, through reductive amination or alkylation of the derived alcohol. In addition, the reaction of ketone 8 with several lithium acetylides allowed the stereoselective synthesis of new azasugars alkylated at C-3. The activity of the new compounds towards lysosomal ß-galactosidase was negligible, showing that the presence of an alkyl chain in this position is detrimental to inhibitory activity. Interestingly, 9, 10, and 12 behave as good inhibitors of lysosomal ß-glucosidase (GCase) (IC50 = 12, 6.4, and 60 µM, respectively). When tested on cell lines bearing the Gaucher mutation, they did not impart any enzyme rescue. However, altogether, the data included in this work give interesting hints for the design of novel inhibitors.
Assuntos
Carboidratos/química , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Cetonas/química , Piperidinas/síntese química , Piperidinas/farmacologia , beta-Galactosidase/antagonistas & inibidores , beta-Glucosidase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Piperidinas/químicaRESUMO
In the era of green economy, trehalase inhibitors represent a valuable chance to develop non-toxic pesticides, being hydrophilic compounds that do not persist in the environment. The lesson on this topic that we learned from the past can be of great help in the research on new specific green pesticides. This review aims to describe the efforts made in the last 50 years in the evaluation of natural compounds and their analogues as trehalase inhibitors, in view of their potential use as insecticides and fungicides. Specifically, we analyzed trehalase inhibitors based on sugars and sugar mimics, focusing on those showing good inhibition properties towards insect trehalases. Despite their attractiveness as a target, up to now there are no trehalase inhibitors that have been developed as commercial insecticides. Although natural complex pseudo di- and trisaccharides were firstly studied to this aim, iminosugars look to be more promising, showing an excellent specificity profile towards insect trehalases. The results reported here represent an overview and a discussion of the best candidates which may lead to the development of an effective insecticide in the future.
Assuntos
Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Insetos/antagonistas & inibidores , Inseticidas/farmacologia , Trealase/antagonistas & inibidores , Animais , Humanos , SegurançaRESUMO
The synthesis of selective inhibitors of human carbonic anhydrases (hCAs) is of paramount importance to avoid side effects derived from undesired interactions with isoforms not involved in the targeted pathology, and this was partially addressed with the introduction of a sugar moiety (the so-called "sugar approach"). Since glycomimetics are considered more selective than the parent sugars in inhibiting carbohydrate-processing enzyme, we explored the possibility of further tuning the selectivity of hCAs inhibitors by combining the sulfonamide moiety with a sugar analogue residue. In particular, we report the synthesis of two novel hCAs inhibitors 2 and 3 which feature the presence of a piperidine iminosugar and an additional carbohydrate moiety derived from levoglucosenone (1), a key intermediate derived from cellulose pyrolysis. Biological assays revealed that iminosugar 2 is a very strong inhibitor of the central nervous system (CNS) abundantly expressed hCA VII (K I of 7.4 nM) and showed a remarkable selectivity profile toward this isoform. Interestingly, the presence of levoglucosenone in glycomimetic 3 imparted a strong inhibitory activity toward the tumor associated hCA IX (K I of 35.9 nM).
RESUMO
Gaucher disease is caused by mutations in human acid ß-glucosidase or glucocerebrosidase (GCase), the enzyme responsible for hydrolysis of glucosyl ceramide in the lysosomes. Imino- and azasugars such as 1-deoxynojirimycin and isofagomine are strong inhibitors of the enzyme and are of interest in pharmacological chaperone therapy of the disease. Despite several crystal structures of the enzyme with the imino- and azasugars bound in the active site having been resolved, the actual acid-base chemistry of the binding is not known. In this study we show, using photoinduced electron transfer (PET), that 1-deoxynojirimycin and isofagomine derivatives are protonated by human acid ß-glucosidase when bound, even if they are completely unprotonated outside the enzyme. While isofagomine derivative protonation to some degree was foreshadowed by earlier crystal structures, 1-deoxynojirimycin derivatives were not believed to act as basic amines in the enzyme.