RESUMO
In the design of inhibitors of phosphosugar metabolizing enzymes and receptors with therapeutic interest, malonate has been reported in a number of cases as a good and hydrolytically-stable surrogate of the phosphate group, since both functions are dianionic at physiological pH and of comparable size. We have investigated a series of malonate-based mimics of the best known phosphate inhibitors of class II (zinc) fructose-1,6-bis-phosphate aldolases (FBAs) (e.g., from Mycobacterium tuberculosis), type I (zinc) phosphomannose isomerase (PMI) from Escherichia coli, and phosphoglucose isomerase (PGI) from yeast. In the case of FBAs, replacement of one phosphate by one malonate on a bis-phosphorylated inhibitor (1) led to a new compound (4) still showing a strong inhibition (K(i) in the nM range) and class II versus class I selectivity (up to 8×10(4)). Replacement of the other phosphate however strongly affected binding efficiency and selectivity. In the case of PGI and PMI, 5-deoxy-5-malonate-D-arabinonohydroxamic acid (8) yielded a strong decrease in binding affinities when compared to its phosphorylated parent compound 5-phospho-D-arabinonohydroxamic acid (2). Analysis of the deposited 3D structures of the kinetically evaluated enzymes complexed to the phosphate-based inhibitors indicate that malonate could be a good phosphate surrogate only if phosphate is not tightly bound at the enzyme active site, such as in position 7 of compound 1 for FBAs. These observations are of importance for further design of inhibitors of phosphorylated-compounds metabolizing enzymes with therapeutic interest.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Malonatos/síntese química , Manose-6-Fosfato Isomerase/antagonistas & inibidores , Animais , Domínio Catalítico , Ativação Enzimática/efeitos dos fármacos , Escherichia/enzimologia , Humanos , Concentração Inibidora 50 , Malonatos/química , Malonatos/farmacologia , Modelos Biológicos , Estrutura Molecular , Leveduras/enzimologiaRESUMO
The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/metabolismo , Gluconeogênese , Mycobacterium tuberculosis/enzimologia , Tuberculose Pulmonar/enzimologia , Animais , Proteínas de Bactérias/genética , Cristalografia por Raios X , Fibrinolisina/genética , Fibrinolisina/metabolismo , Frutose-Bifosfato Aldolase/genética , Frutosedifosfatos/química , Frutosedifosfatos/genética , Frutosedifosfatos/metabolismo , Técnicas de Silenciamento de Genes , Cobaias , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Ligação Proteica , Tuberculose Pulmonar/genética , alfa 2-Antiplasmina/genética , alfa 2-Antiplasmina/metabolismoRESUMO
Several 5-O-alkyl- and 5-C-alkyl-mannitol bis-phosphates were synthesized and comparatively assayed as inhibitors of fructose bis-phosphate aldolases (Fbas) from rabbit muscle (taken as surrogate model of the human enzyme) and from Trypanosoma brucei. A limited selectivity was found in several instances. Crystallographic studies confirm that the 5-O-methyl derivative binds competitively with substrate and the 5-O-methyl moiety penetrating deeper into a shallow hydrophobic pocket at the active site. This observation can lead to the preparation of selective competitive or irreversible inhibitors of the parasite Fba.
RESUMO
We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows K(i) against class II Fbas from various pathogens in the nM range, with very high selectivity (up to 10(5)). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.
Assuntos
Antibacterianos/síntese química , Antifúngicos/síntese química , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Ácidos Hidroxâmicos/síntese química , Zinco/fisiologia , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Antituberculosos/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Frutosedifosfatos/química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Concentração Inibidora 50 , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Coelhos , Relação Estrutura-Atividade , Fosfatos Açúcares/químicaRESUMO
We hereby describe the rationale synthesis and biochemical evaluation of the most powerful and selective inhibitors of class II fructose bis-phosphate aldolases so far reported. These inhibitors are of potential therapeutic interest, since the class II enzyme is present exclusively in microorganisms (among which many pathogenic species) and is absent from man, plants, and animals.
RESUMO
We report the synthesis and biochemical evaluation of selective inhibitors of class II (zinc-dependent) fructose bisphosphate aldolases. The most active compound is a simplified analogue of fructose bisphosphate, bearing a well-positioned metal chelating group. It is a powerful and highly selective competitive inhibitor of isolated class II aldolases. We report crystallographic studies of this inhibitor bound in the active site of the Helicobacter pylori enzyme. The compound also shows activity against Mycobacterium tuberculosis isolates.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Cristalografia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacosRESUMO
A pair of novel C3-epimeric sugar-derived ligands (glycoligands) with a neutral N4O donor set was synthesized. Copper(II) complexes of both ligands were obtained and characterized by X-ray crystallography. Cyclic voltammetry, electron paramagnetic resonance, and UV-vis spectroscopies showed similar electronic properties. Mirror-image CD spectra were obtained for the Cu(II) d-d band, indicating an enantiomeric character of the coordination sphere, which has been rationalized structurally. This example shows the possible predetermination of stereochemistry for complexes by ligands based on a glycoscaffold.
Assuntos
Carboidratos/química , Cobre/química , Dicroísmo Circular , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Ligantes , Modelos Moleculares , Estrutura Molecular , EstereoisomerismoRESUMO
We report the first unambiguous syntheses of glucitol-1,6-bis-phosphate and mannitol-1,6-bis-phosphate and their competitive inhibition of various fructose bis-phosphate aldolases.
Assuntos
Frutose-Bifosfato Aldolase/antagonistas & inibidores , Manitol Fosfatos/síntese química , Sorbitol/análogos & derivados , Sorbitol/síntese química , Fosfatos Açúcares/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Helicobacter pylori/enzimologia , Manitol Fosfatos/farmacologia , Estrutura Molecular , Saccharomyces cerevisiae/enzimologia , Sorbitol/farmacologia , Fosfatos Açúcares/farmacologiaRESUMO
Two new ligands of transition metal cations based on galactose-derived scaffolds were synthesised: 1,5-anhydro-2-deoxy-3,4,6-tri-O-(2-picolyl)-D-galactitol and methyl 2-deoxy-3,4,6-tri-O-(2-picolyl)-alpha-D-galactopyranoside. These ligands permitted the isolation as single crystals of a Co(II) and a Ni(II) complex, respectively. The structures of both complexes were determined by X-ray crystallography showing a coordination sphere including sugar-bound oxygen atoms. The sugar-derived ligands were found to be in both cases in high energy conformations in the crystal structures of the complexes. These conformations contain an arrangement of sugar-bound oxygen atoms similar to those observed in polyol-metal and carbohydrate-metal complexes.
Assuntos
Galactitol/química , Galactose/química , Compostos Organometálicos/síntese química , Elementos de Transição , Configuração de Carboidratos , Cristalização , Cristalografia por Raios X , Desoxiaçúcares , Ligantes , Compostos Organometálicos/químicaRESUMO
The title compound, [Co(C(32)H(35)N(3)O(6))(H2O)2](ClO4)(2).H2O, contains a cationic complex with a novel facultative hexadentate sugar-derived ligand coordinated in a tetradentate fashion to give a CoN(2)O(4) coordination. The partial coordination is imposed by the rigid conformation of the sugar. The pyridine group that is not bound to the metal is free to participate in intermolecular hydrogen bonding and pi-pi stacking interactions.
RESUMO
We report the synthesis and biochemical evaluation of new competitive inhibitors of the cytosolic (NADH-dependent) glycerophosphate dehydrogenase. The best tested compound, phosphono-propionohydroxamic acid, with a Ki of 6 microM, might be of interest as an anti-obesity drug.
Assuntos
Citosol/enzimologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glicerolfosfato Desidrogenase/antagonistas & inibidores , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacologia , Músculo Esquelético/enzimologia , NAD/metabolismo , Animais , Citosol/efeitos dos fármacos , Humanos , Ligação de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Conformação Molecular , Músculo Esquelético/efeitos dos fármacos , Coelhos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A SOD-like activity evaluated by a modified McCord-Fridovich test was evidenced for two Co(II) complexes built from "glycoligands" using a sugar platform derived from d-galactose and D-galactal and functionalized by three 2-picolyl groups.
Assuntos
Metabolismo dos Carboidratos , Carboidratos/química , Cobalto/química , Superóxido Dismutase/metabolismo , Cátions/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Especificidade por SubstratoRESUMO
D-glucosaminic acid (2-amino-2-deoxy-D-gluconic acid), a component of bacterial lipopolysaccharides and a chiral synthon, is easily prepared on a multigram scale by air oxidation of D-glucosamine (2-amino-2-deoxy-D-glucose) catalysed by glucose oxidase.
Assuntos
Glucosamina/análogos & derivados , Glucosamina/biossíntese , Glucosamina/metabolismo , Aspergillus niger/enzimologia , Catalase/metabolismo , Glucosamina/química , Glucose Oxidase/metabolismo , Concentração de Íons de Hidrogênio , Estrutura Molecular , OxirreduçãoRESUMO
Degradation of 2,6-dichlorophenol (2,6-DCP) was accomplished by oxidation catalyzed by Coprinus cinereus peroxidase. Immobilization of the enzyme in a polyacrylamide matrix enhanced DCP oxidation. Hydrogen peroxide, peroxidase's natural substrate, was produced enzymatically in situ to avoid peroxidase inactivation by its too high concentration. In the case of larger scale utilization, the method would also avoid direct handling of this hazardous reagent.
Assuntos
Clorofenóis/química , Coprinus/enzimologia , Peróxido de Hidrogênio/síntese química , Peroxidase/química , Catálise , Enzimas Imobilizadas/química , Humanos , OxirreduçãoRESUMO
Phosphoglycolo amidoxime and phosphoglycolo hydrazide, two new derivatives of phosphoglycolic acid, were synthesised and successfully tested as selective competitive inhibitors of class II FBP-aldolases.