RESUMO
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
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
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 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
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
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
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
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 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
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
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
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.
Assuntos
1-Desoxinojirimicina/análogos & derivados , Inibidores Enzimáticos/química , Glucosilceramidase/química , Imino Piranoses/química , Prótons , Ensaios Enzimáticos , Corantes Fluorescentes/química , Glucosilceramidase/antagonistas & inibidores , Humanos , Fenantrenos/químicaRESUMO
The diffusion of type 2 diabetes (T2D) throughout the world represents one of the most important health problems of this century. Patients suffering from this disease can currently be treated with numerous oral anti-hyperglycaemic drugs, but none is capable of reproducing the physiological action of insulin and, in several cases, they induce severe side effects. Developing new anti-diabetic drugs remains one of the most urgent challenges of the pharmaceutical industry. Multi-target drugs could offer new therapeutic opportunities for the treatment of T2D, and the reported data on type 2 diabetic mice models indicate that these drugs could be more effective and have fewer side effects than mono-target drugs. α-Glucosidases and Protein Tyrosine Phosphatase 1B (PTP1B) are considered important targets for the treatment of T2D: the first digest oligo- and disaccharides in the gut, while the latter regulates the insulin-signaling pathway. With the aim of generating new drugs able to target both enzymes, we synthesized a series of bifunctional compounds bearing both a nitro aromatic group and an iminosugar moiety. The results of tests carried out both in vitro and in a cell-based model, show that these bifunctional compounds maintain activity on both target enzymes and, more importantly, show a good insulin-mimetic activity, increasing phosphorylation levels of Akt in the absence of insulin stimulation. These compounds could be used to develop a new generation of anti-hyperglycemic drugs useful for the treatment of patients affected by T2D.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucosidases/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Imino Açúcares/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Diabetes Mellitus Tipo 2/metabolismo , Relação Dose-Resposta a Droga , Glucosidases/metabolismo , Células Hep G2 , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Imino Açúcares/síntese química , Imino Açúcares/química , Conformação Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Pirrolidinas/metabolismo , alfa-Manosidase/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Drosophila melanogaster/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Imino Açúcares/síntese química , Imino Açúcares/química , Imino Açúcares/metabolismo , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Pirrolidinas/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , alfa-Manosidase/antagonistas & inibidores , alfa-Manosidase/genéticaRESUMO
Hyacinthacines are members of the class of polyhydroxylated pyrrolizidines exhibiting outstanding biological activity as glycosidases inhibitors. Their structural complexity is embodied in the densely functionalized core, possessing a series of contiguous stereogenic centers. In this synthetic study we report a route to the more complex congeners of this class of alkaloids exploiting the diastereoselective addition of an axially chiral lithiated alkoxyallene to an enantiopure cyclic nitrone. Our stereodivergent approach enabled the installation of the targeted configuration at the ring A by minimal synthetic manipulations and at ring B by using stage dependent selective functionalizations. The versatility and robustness of this methodology were demonstrated by the syntheses of (-)-hyacinthacine B4 and of two epimers of (+)-hyacinthacine C5, allowing a suggestion of the likely structure of the isolated natural product.
Assuntos
Aldeídos/química , Alcenos/química , Alcaloides de Pirrolizidina/síntese química , Conformação Molecular , Alcaloides de Pirrolizidina/química , EstereoisomerismoRESUMO
A collection of carbohydrate-derived iminosugars belonging to three structurally diversified sub-classes (polyhydroxylated pyrrolidines, piperidines, and pyrrolizidines) was evaluated for inhibition of human acid ß-glucosidase (glucocerebrosidase, GCase), the deficient enzyme in Gaucher disease. The synthesis of several new pyrrolidine analogues substituted at the nitrogen or α-carbon atom with alkyl chains of different lengths suggested an interpretation of the inhibition data and led to the discovery of two new GCase inhibitors at sub-micromolar concentration. In the piperidine iminosugar series, two N-alkylated derivatives were found to rescue the residual GCase activity in N370S/RecNcil mutated human fibroblasts (among which one up to 1.5-fold). This study provides the starting point for the identification of new compounds in the treatment of Gaucher disease.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Doença de Gaucher/enzimologia , Glucosilceramidase/antagonistas & inibidores , Imino Açúcares/química , Imino Açúcares/farmacologia , Descoberta de Drogas , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/metabolismo , HumanosRESUMO
The synthesis of new multivalent architectures based on a trihydroxypiperidine α-fucosidase inhibitor is reported herein. Tetravalent and nonavalent dendrimers were obtained by means of the click chemistry approach involving the copper azide-alkyne-catalyzed cycloaddition (CuAAC) between suitable scaffolds bearing terminal alkyne moieties and an azido-functionalized piperidine as the bioactive moiety. A preliminary biological investigation is also reported towards commercially available and human glycosidases.
RESUMO
The synthesis of the first multivalent pyrrolizidine iminosugars is reported. The key azido intermediates 4 and 31 were prepared after suitable synthetic elaboration of the cycloadduct obtained from 1,3-dipolar cycloaddition of D-arabinose derived nitrone to dimethylacrylamide. The key step of the strategy was the stereoselective installation of an azido moiety at C-6 of the pyrrolizidine skeleton. The click reaction with different monovalent and dendrimeric alkyne scaffolds allowed the preparation of a library of new mono- and multivalent pyrrolizidine compounds that were preliminarily assayed as glycosidase inhibitors towards a panel of commercially available glycosyl hydrolases.
Assuntos
Pirróis/química , Alcaloides de Pirrolizidina/química , Reação de Cicloadição , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Pirróis/síntese química , Alcaloides de Pirrolizidina/síntese química , Saccharomyces cerevisiae/enzimologiaRESUMO
Addition of lithiated 1-benzyloxyallene to a D-arabinose-derived cyclic nitrone occurred with perfect diastereoselectivity furnishing a bicyclic 1,2-oxazine derivative, which is an excellent precursor for pyrrolizidine alkaloids hydroxylated at C-7 with optional configuration at this stereogenic center. Depending on the stage of the N-O bond cleavage and ring re-closure, 7-hydroxypyrrolizidines with 7R or 7S configuration were obtained, as a result of completely selective addition reactions occurring complementarily at the bottom or top face of the endocyclic C-C double bond in six- and five-membered B rings, respectively. Applicability of these stereodivergent routes to obtain polyhydroxy pyrrolizidine alkaloids is demonstrated by the efficient syntheses of casuarine and australine as examples of the two classes of diversely configured 7-hydroxypyrrolizidine alkaloids. An alternative synthesis of australine and two strategies for the preparation of 7-epi-australine are also reported, which demonstrate that the stereoselectivity of hydride reduction of an exocyclic C-O double bond is independent of the ring size, occurring preferentially from the top face either in a six- or five-membered ring.