RESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Glucosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Malonatos/síntesis química , Manosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Animales , Dominio Catalítico , Activación Enzimática/efectos de los fármacos , Escherichia/enzimología , Humanos , Concentración 50 Inhibidora , Malonatos/química , Malonatos/farmacología , Modelos Biológicos , Estructura Molecular , Levaduras/enzimologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Fructosa-Bifosfato Aldolasa/química , Fructosa-Bifosfato Aldolasa/metabolismo , Gluconeogénesis , Mycobacterium tuberculosis/enzimología , Tuberculosis Pulmonar/enzimología , Animales , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Fibrinolisina/genética , Fibrinolisina/metabolismo , Fructosa-Bifosfato Aldolasa/genética , Fructosadifosfatos/química , Fructosadifosfatos/genética , Fructosadifosfatos/metabolismo , Técnicas de Silenciamiento del Gen , Cobayas , Interacciones Huésped-Patógeno/genética , Humanos , Ratones , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Unión Proteica , Tuberculosis Pulmonar/genética , alfa 2-Antiplasmina/genética , alfa 2-Antiplasmina/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Antibacterianos/síntesis química , Antifúngicos/síntesis química , Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Ácidos Hidroxámicos/síntesis química , Zinc/fisiología , Animales , Antibacterianos/química , Antibacterianos/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Antituberculosos/síntesis química , Antituberculosos/química , Antituberculosos/farmacología , Cristalografía por Rayos X , Diseño de Fármacos , Fructosadifosfatos/química , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Concentración 50 Inhibidora , Cinética , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conejos , Relación Estructura-Actividad , Fosfatos de Azúcar/químicaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Cristalografía , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/efectos de los fármacosRESUMEN
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.
Asunto(s)
Carbohidratos/química , Cobre/química , Dicroismo Circular , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Ligandos , Modelos Moleculares , Estructura Molecular , EstereoisomerismoRESUMEN
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.
Asunto(s)
Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Manitol Fosfatos/síntesis química , Sorbitol/análogos & derivados , Sorbitol/síntesis química , Fosfatos de Azúcar/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Helicobacter pylori/enzimología , Manitol Fosfatos/farmacología , Estructura Molecular , Saccharomyces cerevisiae/enzimología , Sorbitol/farmacología , Fosfatos de Azúcar/farmacologíaRESUMEN
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.
Asunto(s)
Galactitol/química , Galactosa/química , Compuestos Organometálicos/síntesis química , Elementos de Transición , Conformación de Carbohidratos , Cristalización , Cristalografía por Rayos X , Desoxiazúcares , Ligandos , Compuestos Organometálicos/químicaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Citosol/enzimología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glicerolfosfato Deshidrogenasa/antagonistas & inhibidores , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacología , Músculo Esquelético/enzimología , NAD/metabolismo , Animales , Citosol/efectos de los fármacos , Humanos , Enlace de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Conformación Molecular , Músculo Esquelético/efectos de los fármacos , Conejos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
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.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Carbohidratos/química , Cobalto/química , Superóxido Dismutasa/metabolismo , Cationes/química , Espectroscopía de Resonancia Magnética , Estructura Molecular , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Glucosamina/análogos & derivados , Glucosamina/biosíntesis , Glucosamina/metabolismo , Aspergillus niger/enzimología , Catalasa/metabolismo , Glucosamina/química , Glucosa Oxidasa/metabolismo , Concentración de Iones de Hidrógeno , Estructura Molecular , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
Clorofenoles/química , Coprinus/enzimología , Peróxido de Hidrógeno/síntesis química , Peroxidasa/química , Catálisis , Enzimas Inmovilizadas/química , Humanos , Oxidación-ReducciónRESUMEN
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.