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1.
Org Biomol Chem ; 14(44): 10371-10385, 2016 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-27735004

RESUMO

This paper explores the computer modelling aided design and synthesis of ß-N-acetylhexosaminidase inhibitors along with their applicability to human disease treatment through biological evaluation in both an enzymatic and cellular setting. We investigated the importance of individual stereocenters, variations in structure-activity relationships along with factors influencing cell penetration. To achieve these goals we modified nitrogen heterocycles in terms of ring size, side chains present and ring nitrogen derivatization. By reducing the inhibitor interactions with the active site down to the essentials we were able to determine that besides the established 2S,3R trans-relationship, the presence and stereochemistry of the CH2OH side chain is of crucial importance for activity. In terms of cellular penetration, N-butyl side chains favour cellar uptake, while hydroxy- and carboxy-group bearing sidechains on the ring nitrogen retarded cellular penetration. Furthermore we show an early proof of principle study that ß-N-acetylhexosaminidase inhibitors can be applicable to use in a potential anti-invasive anti-cancer strategy.

2.
Org Biomol Chem ; 12(44): 8977-96, 2014 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-25277226

RESUMO

The glycosidase inhibitory properties of synthetic C-alkyl and N-alkyl six-membered iminosugars have been extensively studied leading to therapeutic candidates. The related seven-membered iminocyclitols have been less examined despite the report of promising structures. Using an in house ring enlargement/C-alkylation as well as cross-metathesis methodologies as the key steps, we have undertaken the synthesis and biological evaluation of a library of fourteen 2C- and eight N-alkyl tetrahydroxylated azepanes starting from an easily available glucopyranose-derived azidolactol. Four, six, nine and twelve carbon atom alkyl chains have been introduced. The study of two distinct D-gluco and L-ido stereochemistries for the tetrol pattern as well as R and S configurations for the C-2 carbon bearing the C-alkyl chain is reported. We observed that C-alkylation of the L-ido tetrahydroxylated azepane converts it from an α-L-fucosidase to a ß-glucosidase and ß-galactosidase inhibitor while N-alkylation of the D-gluco iminosugar significantly improves its inhibition profile leading to potent ß-glucosidase, ß-galactosidase, α-L-rhamnosidase and ß-glucuronidase inhibitors whatever the stereochemistry of the alkyl chain. Interestingly, the N-alkyl chain length usually parallels the azepane inhibitor potency as exemplified by the identification of a potent glucocerebrosidase inhibitor (Ki 1 µM) bearing a twelve carbon atom chain. Additionally, several C-alkyl azepanes demonstrated promising F508del-CFTR correction unlike the parent tetrahydroxyazepanes. None of the C-alkyl and N-alkyl azepanes did inhibit ER α-glucosidases I or II.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glucosilceramidase/antagonistas & inibidores , Imino Açúcares/farmacologia , Alquilação , Cristalografia por Raios X , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glucosilceramidase/metabolismo , Humanos , Imino Açúcares/síntese química , Imino Açúcares/química , Modelos Moleculares , Conformação Molecular , Relação Estrutura-Atividade
3.
Acta Crystallogr Sect E Struct Rep Online ; 66(Pt 2): o404, 2010 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-21579824

RESUMO

X-Ray crystallography unequivocally determined the stereochemistry of the thymine base in the title compound, C(14)H(18)N(2)O(7). The absolute stereochemistry was determined from the use of d-ribose as the starting material. There are two independent mol-ecules in the asymmetric unit (Z' = 2) which exist as N-H⋯O hydrogen-bonded pairs in the crystal structure.

4.
Acta Crystallogr Sect E Struct Rep Online ; 66(Pt 2): o406-7, 2010 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-21579826

RESUMO

X-ray crystallography unequivocally confirmed the stereochemistry of the 2-C-methyl group in the title mol-ecule, C(10)H(16)O(6), in which the 1,5-lactone ring exists in a boat conformation. The use of d-galactose in the synthesis determined the absolute stereochemistry. The crystal exists as O-H⋯O hydrogen-bonded layers in the ab plane, with each mol-ecule acting as a donor and acceptor for two hydrogen bonds.

5.
Acta Crystallogr Sect E Struct Rep Online ; 66(Pt 4): o853, 2010 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-21580677

RESUMO

The crystal structure of the title compound, C(12)H(18)N(2)O(8), exists as O-H⋯O hydrogen-bonded layers of mol-ecules running parallel to the ab plane. Each mol-ecule is a donor and acceptor for six hydrogen bonds. The absolute stereochemistry was determined by the use of d-glucuronolactone as the starting material.

6.
Acta Crystallogr Sect E Struct Rep Online ; 67(Pt 1): o38-9, 2010 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-21522748

RESUMO

Reaction of 3-O-benzyl-1,2-O-isopropyl-idene-α-xylo-pentodialdo-1,4-furan-ose with N,N-diethyl-2-(dimethyl-sulfuranil-idene)acetamide gave stereoselectively an ep-oxy-amide, which was regioselectively opened by NaN(3) in dimethyl formamide to give the title compound, C(21)H(30)N(4)O(6). X-ray crystallography confirmed the relative stereochemistry of the title compound and the absolute configuration was determined by the use of d-glucose as the starting material. There are two mol-ecules in the asymmetric unit (Z' = 2). The crystal structure consists of two types of chains of O-H⋯O hydrogen-bonded mol-ecules running parallel to the b axis, with each mol-ecule acting as a donor and acceptor of one hydrogen bond.

7.
Phytochemistry ; 56(3): 265-95, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11243453

RESUMO

Over one hundred polyhydroxylated alkaloids have been isolated from plants and micro-organisms. These alkaloids can be potent and highly selective glycosidase inhibitors and are arousing great interest as tools to study cellular recognition and as potential therapeutic agents. However, only three of the natural products so far have been widely studied for therapeutic potential due largely to the limited commercial availability of the other compounds.


Assuntos
Alcaloides/química , Alcaloides/uso terapêutico , Alcaloides/toxicidade , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Inibidores Enzimáticos/toxicidade , Glicosídeo Hidrolases/antagonistas & inibidores , Testes de Toxicidade
8.
Eur J Biochem ; 268(1): 35-41, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11121099

RESUMO

The extract of bark of Angylocalyx pynaertii (Leguminosae) was found to potently inhibit mammalian alpha-L-fucosidases. A thorough examination of the extract resulted in the discovery of 15 polyhydroxylated alkaloids, including the known alkaloids from seeds of this plant, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), 1-deoxymannojirimycin (DMJ) and 2,5-imino-1,2,5-trideoxy-D-mannitol (6-deoxy-DMDP). Among them, eight sugar-mimic alkaloids showed the potent inhibitory activity towards bovine epididymis alpha-L-fucosidase and their Ki values are as follows: 6-deoxy-DMDP (83 microM), 2,5-imino-1,2,5-trideoxy-L-glucitol (0.49 microM), 2,5-dideoxy-2,5-imino-D-fucitol (17 microM), 2,5-imino-1,2,5-trideoxy-D-altritol (3.7 microM), DMJ (4.7 microM), N-methyl-DMJ (30 microM), 6-O-alpha-L-rhamnopyranosyl-DMJ (Rha-DMJ, 0.06 microM), and beta-L-homofuconojirimycin (beta-HFJ, 0.0053 microM). We definitively deduced the structural requirements of inhibitors of alpha-L-fucosidase for the piperidine alkaloids (DMJ derivatives). The minimum structural feature of alpha-L-fucosidase inhibitors is the correct configuration of the three hydroxyl groups on the piperidine ring corresponding to C2, C3 and C4 of L-fucose. Furthermore, the addition of a methyl group in the correct configuration to the ring carbon atom corresponding to C5 of L-fucose generates extremely powerful inhibition of alpha-L-fucosidase. The replacement of the methyl group of beta-HFJ by a hydroxymethyl group reduced its inhibitory potential about 80-fold. This suggests that there may be a hydrophobic region in or around the active site. The existence or configuration of a substituent group on the ring carbon atom corresponding to the anomeric position of L-fucose does not appear to be important for the inhibition. Interestingly, Rha-DMJ was a 70-fold more potent inhibitor of alpha-L-fucosidase than DMJ. This implies that the lysosomal alpha-L-fucosidase may have subsites recognizing oligosaccharyl structures in natural substrates.


Assuntos
Alcaloides/farmacologia , Inibidores Enzimáticos/farmacologia , Fabaceae/química , Plantas Medicinais , alfa-L-Fucosidase/antagonistas & inibidores , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/farmacologia , Alcaloides/química , Animais , Bovinos , Inibidores Enzimáticos/química , Humanos , Caules de Planta/química , Relação Estrutura-Atividade
9.
Carbohydr Res ; 323(1-4): 73-80, 2000 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-10782288

RESUMO

2,6-Dideoxy-7-O-(beta-D-glucopyranosyl) 2,6-imino-D-glycero-L-gulo- heptitol (7-O-beta-D-glucopyranosyl-alpha-homonojirimycin, 1) was isolated from the 50% methanol extract of the whole plant of Lobelia sessilifolia (Campanulaceae), which was found to potently inhibit rice alpha-glucosidase. Adenophorae radix, roots of Adenophora spp. (Campanulaceae), yielded new homonojirimycin derivatives, adenophorine (2), 1-deoxyadenophorine (3), 5-deoxyadenophorine (4), 1-C-(5-amino-5-deoxy-beta-D-galactopyranosyl)butane (beta-1-C-butyl-deoxygalactonojirimycin, 5), and the 1-O-beta-D-glucosides of 2 (6) and 4 (7), in addition to the recently discovered alpha-1-C-ethylfagomine (8) and the known 1-deoxymannojirimycin (9) and 2R,5R-bis(hydroxymethyl)-3R,4R- dihydroxypyrrolidine (DMDP, 10). Compound 4 is a potent inhibitor of coffee bean alpha-galactosidase (IC50 = 6.4 microM) and a reasonably good inhibitor of bovine liver beta-galactosidase (IC50 = 34 microM). Compound 5 is a very specific and potent inhibitor of coffee bean alpha-galactosidase (IC50 = 0.71 microM). The glucosides 1 and 7 were potent inhibitors of various alpha-glucosidases, with IC50 values ranging from 1 to 0.1 microM. Furthermore, 1 potently inhibited porcine kidney trehalase (IC50 = 0.013 microM) but failed to inhibit alpha-galactosidase, whereas 7 was a potent inhibitor of alpha-galactosidase (IC50 = 1.7 microM) without trehalase inhibitory activity.


Assuntos
Asteraceae/química , Glucosídeos/química , Piperidinas/química , 1-Desoxinojirimicina/análogos & derivados , Alcaloides/química , Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Animais , Bovinos , Glicosídeo Hidrolases/antagonistas & inibidores , Imino Piranoses , Concentração Inibidora 50 , Intestinos/enzimologia , Rim/enzimologia , Fígado/enzimologia , Espectroscopia de Ressonância Magnética , Masculino , Extratos Vegetais/química , Proteínas de Plantas/química , Ratos , Ratos Wistar
10.
Phytochemistry ; 53(3): 379-82, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10703061

RESUMO

Adenophora triphylla var. japonica (Campanulaceae) yielded two new alkaloids, the 6-C-butyl derivative of 2R,5R-bis(hydroxymethyl)-3R,4R-dihydroxypyrrolidine (DMDP) and alpha-1-C-ethyl-fagomine, together with the known alkaloids 1,4-dideoxy-1,4-imino-D-arabinitol, 1-deoxynojirimycin, and 1-deoxymannojirimycin. 6-C-Butyl-DMDP showed inhibitory activity toward almond beta-glucosidase (IC50 = 68 microM), whereas alpha-1-C-ethyl-fagomine inhibited bovine liver beta-galactosidase (IC50 = 29 microM).


Assuntos
Alcaloides/isolamento & purificação , Piperidinas/isolamento & purificação , Plantas/química , Pirrolidinas/isolamento & purificação , Alcaloides/química , Hidroxilação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Piperidinas/química , Pirrolidinas/química
11.
J Pept Sci ; 5(10): 425-41, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10580642

RESUMO

The potential of carbohydrate-like tetrahydrofuran frameworks as building blocks with predictable conformational propensities useful in the design and synthesis of novel peptidomimetic materials which adopt well-defined secondary structures is discussed.


Assuntos
Desenho de Fármacos , Furanos/química , Mimetismo Molecular , Peptídeos/química , Aminoácidos/química , Bioquímica/métodos , Carboidratos/química , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Peptídeos/farmacologia , Estrutura Secundária de Proteína
12.
Carbohydr Res ; 316(1-4): 95-103, 1999 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-10515698

RESUMO

Aqueous ethanol extracts from the immature fruits and stalks of bluebell (Hyacinthoides non-scripta) were subjected to various ion-exchange column chromatographic steps to give 1,4-dideoxy-1,4-imino-D-arabinitol (1),2(R),5(R)-bis(hydroxymethyl)-3(R),4(R)-dihydroxypyrrolidine (DMDP) (2), 6-deoxy-6-C-(2,5-dihydroxyhexyl)-DMDP (3),2,5-dideoxy-2,5-imino-DL-glycero-D-manno-heptitol (homoDMDP)(4),homoDMDP-7-O-apioside (5), homoDMDP-7-O-beta-D-xylopyranoside (6), (1S*,2R*,3R*,5R*,7aR*)-1,2-dihydroxy-3,5- dihydroxymethylpyrrolizidine (7), and (1S*,2R*,3R*,5R*,6R*,7R*,7aR*)-3-hydroxymethyl-5-methyl-1,2,6,7 tetrahydroxypyrrolizidine (8). Bulbs of Scilla campanulata (Hyacinthaceae) yielded (1S*,2R*,3R*,5S*,7aR*)-1,2-dihydroxy-3,5-dihydroxy-methylpyrrol izidine (9) in addition to compounds 1-7. Compounds 3,6,7,8, and 9 are new natural products. Compound 4 is a potent competitive inhibitor with K(i) values of 1.5 microM for Caldocellum saccharolyticum beta-glucosidase and 2.2 microM for bovine liver beta-galactosidase. The 7-O-beta-D xyloside 6 was a stronger competitive inhibitor than 4 of C saccharolyticum beta-glucosidase and rat intestinal lactase, with K(i) values of 0.06 and 0.07 microM, respectively, but a weaker inhibitor of bovine liver beta-galactosidase. Furthermore, compound 4 is also a competitive inhibitor (K(i) = 1.8 microM) of porcine kidney trehalase, but 6 was inactive against this enzyme.


Assuntos
Alcaloides/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Glicosídeo Hidrolases/antagonistas & inibidores , Plantas Tóxicas/química , Alcaloides/química , Alcaloides/farmacologia , Animais , Bovinos , Cromatografia por Troca Iônica , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Frutas/química , Cromatografia Gasosa-Espectrometria de Massas , Inibidores de Glicosídeo Hidrolases , Intestinos/enzimologia , Rim/enzimologia , Lactase , Fígado/enzimologia , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Caules de Planta/química , Pirrolidinas/química , Pirrolidinas/isolamento & purificação , Alcaloides de Pirrolizidina/química , Alcaloides de Pirrolizidina/isolamento & purificação , Alcaloides de Pirrolizidina/farmacologia , Ratos , Pele/enzimologia , Suínos , Trealase/antagonistas & inibidores , beta-Galactosidase/antagonistas & inibidores
13.
Protein Sci ; 8(4): 741-9, 1999 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10211820

RESUMO

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.


Assuntos
Crioprotetores/farmacologia , Fosforilases/metabolismo , Monofosfato de Adenosina/metabolismo , Cristalografia por Raios X , Dimetil Sulfóxido/metabolismo , Glucose/antagonistas & inibidores , Glicóis/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Polietilenoglicóis/metabolismo
14.
J Med Chem ; 41(14): 2565-71, 1998 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-9651160

RESUMO

A series of natural epimers of alpha-homonojirimycin and its N-alkylated derivatives have been prepared to investigate the contribution of the different chiral centers and conformation of the specificity and potency of inhibition of glycosidases. These epimers and N-alkylated derivatives are alpha-homonojirimycin (1), beta-homonojirimycin (2), alpha-homomannojirimycin (3), beta-homomannojirimycin (4), alpha-3,4-di-epi-homonojirimycin (5), beta-4,5-di-epi-homonojirimycin (6), N-methyl-alpha-homonojirimycin (7), and N-butyl-alpha-homonojirimycin (8). Compound 1 was a potent inhibitor of a range of alpha-glucosidases with IC50 values of 1 to 0.01 microM. Compounds 2, 3, and 4 were surprisingly inactive as inhibitors of beta-glucosidase and alpha- and beta-mannosidases but were moderately good as inhibitors of rice and some mammalian alpha-glucosidases. Compound 4 was active in the micromolar range toward all alpha-glucosidases tested. Furthermore, compound 4, which superimposes well on beta-l-fucose, was a 10-fold more effective inhibitor of alpha-l-fucosidase than 1-deoxymannojirimycin (12) and 3, with a Ki value of 0.45 microM. Only compounds 5 and 6 showed inhibitory activity toward alpha- and beta-galactosidases (6with an IC50 value of 6.4 microM against alpha-galactosidase). The high-resolution structure of 1 has been determined by X-ray diffraction and showed a chair conformation with the C1 OH (corresponding to the C6 OH in 1-deoxynojirimycin) predominantly equatorial to the piperidine ring in the crystal structure. This preferred (C1 OH equatorial) conformation was also corroborated by 1H NMR coupling constants. The coupling constants for 7 suggest the axial orientation of the C1 OH, while in 8 the C1 OH axial conformation was not observed. The C1 OH axial conformation appears to be responsible for more potent inhibition toward processing alpha-glucosidase I than alpha-glucosidase II. It has been assumed that the anti-HIV activity of alkaloidal glycosidase inhibitors results from the inhibition of processing alpha-glucosidase I, but 1, 7, and 8 were inactive against HIV-1 replication at 500 microg/mL as measured by inhibition of virus-induced cytopathogenicity in MT-4 cells. In contrast, the EC50 value for N-butyl-1-deoxynojirimycin (11), which also inhibits processing alpha-glucosidase I, was 37 microg/mL. Compound 7 has been shown to be a better inhibitor of alpha-glucosidase I than 1 and 8 both in vitro and in the cell culture system. These data imply that inhibition of HIV by glycosidase inhibitors can be due to factors other than simply inhibition of processing alpha-glucosidase I.


Assuntos
Inibidores Enzimáticos/química , Inibidores de Glicosídeo Hidrolases , Piperidinas/química , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/síntese química , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/farmacologia , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Bovinos , Linhagem Celular Transformada , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Imino Piranoses , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estrutura Molecular , Piperidinas/farmacologia , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Replicação Viral/efeitos dos fármacos
15.
J Nat Prod ; 61(5): 625-8, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9599261

RESUMO

Aqueous methanol extracts from the bulbs of Hyacinthusorientalis were subjected to various ion-exchange column chromatographic steps to give 2(R),5(R)-bis(hydroxymethyl)-3(R),4(R)-dihydroxypyrrolidine (DMDP) (1), 2,5-dideoxy-2,5-imino-dl-glycero-d-manno-heptitol (homoDMDP) (2), 2,5-imino-2,5,6-trideoxy-d-manno-heptitol (6-deoxy-homoDMDP) (3), 2,5-imino-2,5,6-trideoxy-d-gulo-heptitol (4), 1-deoxynojirimycin (5), 1-deoxymannojirimycin (6), alpha-homonojirimycin (7), beta-homonojirimycin (8), alpha-homomannojirimycin (9), beta-homomannojirimycin (10), and 7-O-beta-d-glucopyranosyl-alpha-homonojirimycin (MDL 25,637) (11). The structures of the new natural products 3 and 4 were determined by spectroscopic analysis, including extensive 1D and 2D NMR studies. Compound 2 was found to be a potent inhibitor of bacterial beta-glucosidase, mammalian beta-galactosidases, and mammalian trehalases, while 3 was a potent inhibitor of rice alpha-glucosidase and rat intestinal maltase. Compound 4 was observed to be a good inhibitor of alpha-l-fucosidase.


Assuntos
Alcaloides/isolamento & purificação , Glicosídeo Hidrolases/antagonistas & inibidores , Plantas/química , Alcaloides/farmacologia , Animais , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Oryza/enzimologia , Ratos
16.
Protein Sci ; 7(4): 915-27, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9568898

RESUMO

A glucopyranose spirohydantoin (a pyranose analogue of the potent herbicide, hydantocidin) has been identified as the highest affinity glucose analogue inhibitor of glycogen phosphorylase b (GPb). In order to elucidate the structural features that contribute to the binding, the structures of GPb in the native T state conformation and in complex with glucopyranose spirohydantoin have been determined at 100 K to 2.0 A and 1.8 A resolution, respectively, and refined to crystallographic R values of 0.197 (R[free] 0.248) and 0.182 (R[free] 0.229), respectively. The low temperature structure of GPb is almost identical to that of the previously determined room temperature structure, apart from a decrease in overall atomic temperature factors ((B) room temperature GPb = 34.9 A2; (B) 100 K GPb = 23.4 A2). The glucopyranose spirohydantoin inhibitor (Ki = 3.0 microM) binds at the catalytic site and induces small changes in two key regions of the protein: the 280s loop (residues 281-286) that results in a decrease in mobility of this region, and the 380s loop (residues 377-385) that undergoes more significant shifts in order to optimize contact to the ligand. The hydantoin group, that is responsible for increasing the affinity of the glucose compound by a factor of 10(3), makes only one hydrogen bond to the protein, from one of its NH groups to the main chain oxygen of His377. The other polar groups of the hydantoin group form hydrogen bonds to five water molecules. These waters are involved in extensive networks of hydrogen bonds and appear to be an integral part of the protein structure. Analysis of the water structure at the catalytic site of the native enzyme, shows that five waters are displaced by ligand binding and that there is a significant decrease in mobility of the remaining waters on formation of the GPb-hydantoin complex. The ability of the inhibitor to exploit existing waters, to displace waters and to recruit new waters appears to be important for the high affinity of the inhibitor.


Assuntos
Glucose/química , Hidantoínas/química , Fosforilases/química , Água/química , Animais , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Glucose/análogos & derivados , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Músculos/enzimologia , Ligação Proteica/fisiologia , Conformação Proteica , Coelhos , Temperatura
17.
Protein Sci ; 4(12): 2469-77, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8580837

RESUMO

Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to alpha-D-glucose (Ki = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of beta-D-glucopyranosylamine, N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (Ki = 32 microM) and the a (Ki = 35 microM) forms of the enzyme with respect to glucose 1-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase a and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-1-GlcNAc-IMP complex were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 A resolution. 1-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of alpha-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-alpha-D-glucose complex structure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.


Assuntos
Inibidores Enzimáticos/química , Glucosamina/análogos & derivados , Glucose/metabolismo , Fosforilases/antagonistas & inibidores , Animais , Sítios de Ligação , Ligação Competitiva , Cafeína/farmacologia , Cristalografia por Raios X , Sinergismo Farmacológico , Glucosamina/química , Glucosamina/metabolismo , Glucosamina/farmacologia , Cinética , Modelos Moleculares , Estrutura Molecular , Fosforilases/metabolismo , Coelhos , Ultracentrifugação
18.
Biochem J ; 311 ( Pt 3): 845-52, 1995 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-7487940

RESUMO

A series of glucose-analogue inhibitors of glycogen phosphorylase b (GPb) has been designed, synthesized and investigated in crystallographic binding and kinetic studies. The aim is to produce a compound that may exert more effective control over glycogen metabolism than the parent glucose molecule and which could alleviate hyperglycaemia in Type-II diabetes. N-Acetyl-beta-D-glucopyranosylamine (1-GlcNAc) has a Ki for muscle GPb in crude extracts of 30 microM, 367-fold lower than that of beta-D-glucose [Board, Hadwen and Johnson (1995) Eur. J. Biochem. 228, 753-761]. In the current work, the effects of 1-GlcNAc on the activation states of GP and glycogen synthase (GS) in cell-free preparations and in isolated hepatocytes are reported. In gel-filtered extracts of liver, which lack ATP for kinase activity, 1-GlcNAc produced a rapid and time-dependent inactivation of GP with a subsequent activation of GS. Effects of 1-GlcNAc on both enzymes were stronger than those of glucose, with 0.8 mM 1-GlcNAc being equipotent with 50 mM glucose. At 1 mM, 1-GlcNAc enhanced the dephosphorylation of exogenous GPa by liver extracts (600%) and by muscle extracts (75%). This represents an approximately 500-fold improvement on glucose for the liver activity and 40-fold for the muscle activity. In whole hepatocytes, 1-GlcNAc showed an approximately 5-fold enhancement of glucose effects for GP inactivation but failed to elicit activation of GS. Glucose-induced activation of GS in whole hepatocytes was reversed by subsequent addition of 1-GlcNAc. However, when GS activation was achieved via the adenosine analogue and kinase inhibitor, 5'-iodotubercidin (ITU), subsequent addition of 1-GlcNAc allowed continued activation of GS. Phosphorylation of 1-GlcNAc in rat hepatocytes was established using radiolabelled material. The rate of phosphorylation was 1.60 nmol/min per 10(6) cells at 20 mM 1-GlcNAc but was reduced by the presence of 50 microM ITU (0.775 nmol/min per 10(6) cells). It is suggested that the phosphorylated derivative of 1-GlcNAc formed in hepatocytes is 1-GlcNAc 6-phosphate and that the presence of this species is responsible for the failure of 1-GlcNAc to activate GS. The relative importance of the reduction in concentration of GPa versus increased glucose 6-phosphate levels for activation of GS is discussed.


Assuntos
Acetilglucosamina/farmacologia , Glicogênio Sintase/metabolismo , Fígado/enzimologia , Fosforilases/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Glicogênio Hepático/metabolismo , Masculino , Fosforilação , Ratos , Ratos Wistar
19.
Acta Crystallogr D Biol Crystallogr ; 51(Pt 4): 458-72, 1995 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-15299833

RESUMO

Several inhibitors of the large regulatory enzyme glycogen phosphorylase (GP) have been studied in crystallographic and kinetic experiments. GP catalyses the first step in the phosphorylysis of glycogen to glucose-l-phosphate, which is utilized via glycolysis to provide energy to sustain muscle contraction and in the liver is converted to glucose. alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase form b (GPb, K(i) = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several N-acetyl glucopyranosylamine derivatives have been synthesized and tested in a series of crystallographic and kinetic binding studies with GPb. The structural results of the bound enzyme-ligand complexes have been analysed together with the resulting affinities in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. Comparison of the N-methylacetyl glucopyranosylamine (N-methylamide, K(i) = 0.032 mM) with the analogous beta-methylamide derivative (C-methylamide, K(i) = 0.16 mM) illustrate the importance of forming good hydrogen bonds and obtaining complementarity of van der Waals interactions. These studies also have shown that the binding modes can be unpredictable but may be rationalized with the benefit of structural data and that a buried and mixed polar/non-polar catalytic site poses problems for the systematic addition of functional groups. Together with previous studies of glucose analogue inhibitors of GPb, this work forms the basis of a training set suitable for three-dimensional quantitative structure-activity relationship studies. The molecules in the training set are void of problems and potential errors arising from the alignment and bound conformations of each of the ligands since the coordinates were those determined experimentally from the X-ray crystallographic refined ligand-enzyme complexes. The computational procedure described in this work involves the use of the program GRID to describe the molecular structures and the progam GOLPE to obtain the partial least squares regression model with the highest prediction ability. The GRID/GOLPE procedure performed using 51 glucose analogue inhibitors of GPb has good overall predictivity [standard deviation of error predictions (SDEP) = 0.98 and Q(2) = 0.76] and has shown good agreement with the crystallographic and kinetic results by reliably selecting regions that are known to affect the binding affinity.

20.
Eur J Drug Metab Pharmacokinet ; 19(3): 185-92, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-7867660

RESUMO

alpha-D-glucose is a weak inhibitor (Ki = 1.7 mM) of glycogen phosphorylase (GP) and acts as physiological regulator of hepatic glycogen metabolism; it binds to GP at the catalytic site and stabilizes the inactive T state of the enzyme promoting the action of protein phosphatase 1 and stimulating glycogen synthase. The three-dimensional structures of T state rabbit muscle GPb and the GPb-alpha-D-glucose complex have been exploited in the design of better regulators of GP that could shift the balance between glycogen synthesis and glycogen degradation in favour of the former. Close examination of the catalytic site with alpha-D-glucose bound shows that there is an empty pocket adjacent to the beta-1-C position. beta-D-glucose is a poorer inhibitor (Ki = 7.4 mM) than alpha-D-glucose, but mutarotation has prevented the binding of beta-D-glucose in T state GP crystals. A series of beta-D-glucose analogues has been designed and tested in kinetic and crystallographic experiments. Several compounds have been discovered that have an increased affinity for GP than the parent compound.


Assuntos
Desenho de Fármacos , Glucose/farmacologia , Hipoglicemiantes/farmacologia , Fosforilases/antagonistas & inibidores , Cristalografia , Glucose/análogos & derivados , Estereoisomerismo
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