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1.
Biol Pharm Bull ; 32(9): 1618-20, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19721243

RESUMO

Novel glycogen phosphorylase a (GPa) inhibitors with a phenethylphenylphthalimide skeleton were prepared based on alpha-glucosidase inhibitors and liver X receptor (LXR) antagonists derived from thalidomide. Their structure-activity relationships were analyzed. Some of the compounds thus prepared showed potent inhibitory activity against rabbit muscle GPa with more than 10-fold greater efficacy than a typical GPa inhibitor, 1,4-dideoxy-1,4-imino-D-arabinitol.


Assuntos
Inibidores Enzimáticos/química , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Glicogênio Fosforilase Muscular/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases , Receptores Nucleares Órfãos/antagonistas & inibidores , Ftalimidas/síntese química , Talidomida/análogos & derivados , Talidomida/farmacologia , Animais , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase Hepática/química , Glicogênio Fosforilase Muscular/química , Receptores X do Fígado , Receptores Nucleares Órfãos/química , Ftalimidas/farmacologia , Coelhos , Talidomida/química
2.
Bioorg Med Chem Lett ; 18(14): 4068-71, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18554908

RESUMO

A series of amino acid anthranilamide derivatives identified from a high-throughput screening campaign as novel, potent, and glucose-sensitive inhibitors of human liver glycogen phosphorylase a are described. A solid-phase synthesis using Wang resin was also developed which provided efficient access to a variety of analogues, and resulted in the identification of key structure-activity relationships, and the discovery of a potent exemplar (IC(50)=80 nM). The SAR scope, synthetic strategy, and in vitro results for this series are presented herein.


Assuntos
Glicogênio Fosforilase Hepática/antagonistas & inibidores , ortoaminobenzoatos/química , Aminoácidos/química , Animais , Química Farmacêutica/métodos , Desenho de Fármacos , Glicogênio Fosforilase Hepática/química , Humanos , Concentração Inibidora 50 , Fígado/enzimologia , Microssomos Hepáticos/enzimologia , Modelos Químicos , Ratos , Relação Estrutura-Atividade , Ureia/química , ortoaminobenzoatos/farmacologia
3.
Bioorg Med Chem ; 16(10): 5452-64, 2008 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-18434170

RESUMO

A series of 5-chloro-N-aryl-1H-indole-2-carboxamide derivatives were prepared and evaluated as inhibitors of human liver glycogen phosphorylase a (hLGPa). One compound, 5-chloro-N-[4-(1,2-dihydroxyethyl)phenyl]-1H-indole-2-carboxamide (2f), inhibited hLGPa with an IC(50) of 0.90microM. The pyridine analogue of 2f showed inhibitory activity of glucagon-induced glucose output in cultured primary hepatocytes with an IC(50) of 0.62microM and oral hypoglycemic activity in diabetic db/db mice. Crystallographic determination of the complex of 2f with hLGPa showed binding of the inhibitor in a solvent cavity at the dimer interface, with the two hydroxyl groups making favorable electrostatic interactions with hLGPa.


Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Indóis/síntese química , Indóis/farmacologia , Fígado/enzimologia , Administração Oral , Animais , Cristalografia por Raios X , Diabetes Mellitus Experimental/tratamento farmacológico , Inibidores Enzimáticos/química , Glicogênio Fosforilase Hepática/química , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Hipoglicemiantes/uso terapêutico , Indóis/química , Concentração Inibidora 50 , Camundongos , Camundongos Obesos , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo
4.
J Biol Chem ; 283(14): 8913-8, 2008 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-18198182

RESUMO

Disrupting the interaction between glycogen phosphorylase and the glycogen targeting subunit (G(L)) of protein phosphatase 1 is emerging as a novel target for the treatment of type 2 diabetes. To elucidate the molecular basis of binding, we have determined the crystal structure of liver phosphorylase bound to a G(L)-derived peptide. The structure reveals the C terminus of G(L) binding in a hydrophobically collapsed conformation to the allosteric regulator-binding site at the phosphorylase dimer interface. G(L) mimics interactions that are otherwise employed by the activator AMP. Functional studies show that G(L) binds tighter than AMP and confirm that the C-terminal Tyr-Tyr motif is the major determinant for G(L) binding potency. Our study validates the G(L)-phosphorylase interface as a novel target for small molecule interaction.


Assuntos
Glicogênio Fosforilase Hepática/química , Peptídeos/química , Proteína Fosfatase 1/química , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Motivos de Aminoácidos/fisiologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/enzimologia , Dimerização , Glicogênio Fosforilase Hepática/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/metabolismo , Ligação Proteica/fisiologia , Proteína Fosfatase 1/metabolismo , Estrutura Quaternária de Proteína/fisiologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
5.
FEBS Lett ; 581(24): 4749-53, 2007 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-17870073

RESUMO

The inhibition of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) by glycogen phosphorylase a prevents the dephosphorylation and activation of glycogen synthase, suppressing glycogen synthesis when glycogenolysis is activated. Here, we show that a peptide ((280)LGPYY(284)) comprising the last five amino acids of G(L) retains high-affinity interaction with phosphorylase a and that the two tyrosines play crucial roles. Tyr284 deletion abolishes binding of phosphorylase a to G(L) and replacement by phenylalanine is insufficient to restore high-affinity binding. We show that a phosphorylase inhibitor blocks the interaction of phosphorylase a with the G(L) C-terminus, suggesting that the latter interaction could be targeted to develop an anti-diabetic drug.


Assuntos
Glicogênio Fosforilase Hepática/metabolismo , Indóis/farmacologia , Fenilbutiratos/farmacologia , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Calorimetria , Glicogênio Fosforilase Hepática/química , Glicogênio Fosforilase Hepática/genética , Humanos , Camundongos , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Coelhos , Ratos , Termodinâmica , Titulometria , Tirosina/genética
6.
J Inherit Metab Dis ; 30(5): 722-34, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17705025

RESUMO

Deficiency of liver glycogen phosphorylase in glycogen storage disease (GSD) type VI results in a reduced ability to mobilize glucose from glycogen. Six mutations of the PYGL gene, which encodes the liver isoform of the enzyme, have been identified in the literature. We have characterized eight patients from seven families with GSD type VI and identified 11 novel PYGL gene defects. The majority of the mutations were missense, resulting in the substitution of highly conserved residues. These could be grouped into those that were predicted to affect substrate binding (p.V456M, p.E673K, p.S675L, p.S675T), pyridoxal phosphate binding (p.R491C, p.K681T), or activation of glycogen phosphorylase (p.Q13P) or that had an unknown effect (p.N632I and p.D634H). Two mutations were predicted to result in null alleles, p.R399X and [c.1964_1969inv6;c.1969+1_+4delGTAC]. Only 7 of the 23 (30%) reported PYGL alleles carry nonsense, splice site or frameshift mutations compared to 68-80% of affected alleles of the highly homologous muscle glycogen phosphorylase gene, PYGM, that underlie McArdle disease. There was heterogeneity in the clinical symptoms observed in affected individuals. These varied from hepatomegaly and subclinical hypoglycaemia, to severe hepatomegaly with recurrent severe hypoglycaemia and postprandial lactic acidosis. We conclude that deficiency of liver glycogen phosphorylase is predominantly the result of missense mutations affecting enzyme activity. There are no common mutations and the severity of clinical symptoms varies significantly.


Assuntos
Glicogênio Fosforilase Hepática/genética , Doença de Depósito de Glicogênio Tipo IV/genética , Fígado/enzimologia , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Animais , Glicemia/metabolismo , Pré-Escolar , Análise Mutacional de DNA , Éxons , Feminino , Predisposição Genética para Doença , Genótipo , Glicogênio Fosforilase Hepática/química , Glicogênio Fosforilase Hepática/deficiência , Doença de Depósito de Glicogênio Tipo IV/enzimologia , Humanos , Lactente , Íntrons , Ácido Láctico/sangue , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Fenótipo , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Índice de Gravidade de Doença
7.
J Mol Recognit ; 19(5): 451-7, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16691548

RESUMO

Allosteric enzymes have very complex kinetic behaviours which are primarily interpreted through simplified models. To describe the functional properties of liver and muscle glycogen phosphorylase isozymes we have developed an experimental strategy based on the measurements of initial reaction rates in the presence of different concentrations of the effectors glucose-1-phosphate and methyl-xanthines. Using the extensive structural information available for the two glycogen phosphorylase conformers T (inactive) and R (active) with different ligands, we have applied the Monod-Wyman-Changeux model and analysed the results in the context of the exclusive binding of the inhibitors to the T state, meanwhile the substrate glucose-1-phosphate binds to both, the R and T states. The kinetic analysis shows a good agreement between our model and the results obtained from the glycogen phosphorylases and inhibitors included in this study, which demonstrates the validity of the approach described here.


Assuntos
Sítio Alostérico , Glicogênio Fosforilase Hepática/química , Glicogênio Fosforilase Hepática/metabolismo , Glicogênio Fosforilase Muscular/química , Glicogênio Fosforilase Muscular/metabolismo , Regulação Alostérica , Animais , Cafeína/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Coelhos , Xantinas/metabolismo
8.
J Pharmacol Exp Ther ; 317(3): 1230-7, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16537796

RESUMO

In drug discovery, establishing a correlation between in vitro potency and in vivo activity is critical for the validation of the selected target and for developing confidence in the in vitro screening strategy. The present study developed a competition equilibrium dialysis assay using a 96-well dialysis technique to determine the intrinsic Kd for 13 inhibitors of human liver glycogen phosphorylase a (GPa) in the presence of liver homogenate to mimic the physiological environment. The results provided evidence that binding of an inhibitor to GPa was affected by extra cofactors present in the liver homogenate. A good correlation was demonstrated between the in vitro Kd determined under liver homogenate environment and free liver concentration of an inhibitor at the minimum efficacious dose in diabetic ob/ob mice. This study revealed important elements (such as endogenous cofactors missing from the in vitro assay and free concentration at the target tissue) that contributed to a better understanding of the linkage between in vitro and in vivo activity. The approach developed here may be applied to many drugs in pharmacology studies in which the correlation between in vitro and in vivo activities for the target tissue (such as solid tumors, brain, and liver) is critical.


Assuntos
Diabetes Mellitus Experimental , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Fígado/efeitos dos fármacos , Animais , Glicemia/metabolismo , Proteínas Sanguíneas/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/metabolismo , Glicogênio Fosforilase Hepática/química , Humanos , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos , Modelos Biológicos , Ligação Proteica
9.
J Med Chem ; 48(20): 6178-93, 2005 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-16190745

RESUMO

Using a focused screening approach, acyl ureas have been discovered as a new class of inhibitors of human liver glycogen phosphorylase (hlGPa). The X-ray structure of screening hit 1 (IC50 = 2 microM) in a complex with rabbit muscle glycogen phosphorylase b reveals that 1 binds at the AMP site, the main allosteric effector site of the dimeric enzyme. A first cycle of chemical optimization supported by X-ray structural data yielded derivative 21, which inhibited hlGPa with an IC50 of 23 +/- 1 nM, but showed only moderate cellular activity in isolated rat hepatocytes (IC50 = 6.2 microM). Further optimization was guided by (i) a 3D pharmacophore model that was derived from a training set of 24 compounds and revealed the key chemical features for the biological activity and (ii) the 1.9 angstroms crystal structure of 21 in complex with hlGPa. A second set of compounds was synthesized and led to 42 with improved cellular activity (hlGPa IC50 = 53 +/- 1 nM; hepatocyte IC50 = 380 nM). Administration of 42 to anaesthetized Wistar rats caused a significant reduction of the glucagon-induced hyperglycemic peak. These findings are consistent with the inhibition of hepatic glycogenolysis and support the use of acyl ureas for the treatment of type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/síntese química , Monofosfato de Adenosina/química , Sítio Alostérico , Animais , Sítios de Ligação , Cristalografia por Raios X , Glicogênio Fosforilase Hepática/química , Glicogênio Fosforilase Muscular/química , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Técnicas In Vitro , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Coelhos , Ratos , Ureia/química
10.
Protein Sci ; 14(7): 1760-71, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15987904

RESUMO

Acyl ureas were discovered as a novel class of inhibitors for glycogen phosphorylase, a molecular target to control hyperglycemia in type 2 diabetics. This series is exemplified by 6-{2,6-Dichloro- 4-[3-(2-chloro-benzoyl)-ureido]-phenoxy}-hexanoic acid, which inhibits human liver glycogen phosphorylase a with an IC(50) of 2.0 microM. Here we analyze four crystal structures of acyl urea derivatives in complex with rabbit muscle glycogen phosphorylase b to elucidate the mechanism of inhibition of these inhibitors. The structures were determined and refined to 2.26 Angstroms resolution and demonstrate that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Acyl ureas induce conformational changes in the vicinity of the allosteric site. Our findings suggest that acyl ureas inhibit glycogen phosphorylase by direct inhibition of AMP binding and by indirect inhibition of substrate binding through stabilization of the T' state.


Assuntos
Inibidores Enzimáticos/metabolismo , Glicogênio Fosforilase Muscular/antagonistas & inibidores , Músculos/enzimologia , Conformação Proteica/efeitos dos fármacos , Ureia/metabolismo , Monofosfato de Adenosina/metabolismo , Sítio Alostérico , Animais , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Glicogênio Fosforilase Hepática/química , Glicogênio Fosforilase Hepática/metabolismo , Glicogênio Fosforilase Muscular/química , Glicogênio Fosforilase Muscular/metabolismo , Humanos , Hipoglicemiantes , Cinética , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Coelhos , Ureia/análogos & derivados , Ureia/farmacologia
11.
J Mol Graph Model ; 23(5): 457-64, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15781188

RESUMO

Molecular modeling has been used to assist in the development of a novel series of potent glycogen phosphorylase inhibitors based on a phenyl diacid lead, compound 1. In the absence of suitable competitive binding assays, compound 1 was predicted to bind at the AMP allosteric site based on superposition onto known inhibitors which bind at different sites in the enzyme and analyses of the surrounding protein environment associated with these distinct sites. Possible docking modes of compound 1 at the AMP allosteric site were further explored using the crystal structure of rabbit muscle glycogen phosphorylase complexed with a Bayer diacid compound W1807 (PDB entry 3AMV). Compound 1 was predicted to interact with positively charged arginines at the AMP allosteric site in the docking model. Characterization of the binding pocket by a grid-based surface calculation of the docking model revealed a large unfilled hydrophobic region near the central phenyl ring, suggesting that compounds with larger hydrophobic groups in this region would improve binding. A series of naphthyl diacid compounds were designed and synthesized to access this hydrophobic cleft, and showed significantly improved potency.


Assuntos
Desenho Assistido por Computador , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Monofosfato de Adenosina/metabolismo , Sítio Alostérico , Glicogênio Fosforilase/química , Glicogênio Fosforilase/metabolismo , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Glicogênio Fosforilase Hepática/química , Glicogênio Fosforilase Hepática/metabolismo , Glicogênio Fosforilase Muscular/antagonistas & inibidores , Glicogênio Fosforilase Muscular/química , Glicogênio Fosforilase Muscular/metabolismo , Humanos , Técnicas In Vitro , Chumbo/química , Chumbo/farmacologia , Modelos Químicos , Estrutura Molecular , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Termodinâmica
12.
Mol Genet Metab ; 79(2): 142-5, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12809646

RESUMO

We identified a novel mutation in the glycogen phosphorylase gene (PGYL) in a Chinese patient with glycogen storage disease (GSD) type VI. The patient presented with gross hepatomegaly since the age of two without history of any hypoglycemic attack. Otherwise, he was largely asymptomatic. Liver tissue enzyme assays revealed a mild deficiency of total glycogen phosphorylase. Both PGYL and PHKA2 genes were sequenced. The patient was homozygous of a missense mutation G233D in PGYL. This location forms a hairpin turn secondary structure and the small glycine residue is completely conserved in all the orthologous proteins from Escherichia coli to mammals. This is the sixth reported mutation of this form of GSD.


Assuntos
Glicogênio Fosforilase Hepática/genética , Glicogênio Fosforilase Hepática/metabolismo , Doença de Depósito de Glicogênio Tipo VI/genética , Mutação , Sequência de Aminoácidos , Criança , Sequência Conservada , Éxons , Glicogênio Fosforilase Hepática/química , Doença de Depósito de Glicogênio Tipo VI/metabolismo , Doença de Depósito de Glicogênio Tipo VI/patologia , Hepatomegalia/genética , Hepatomegalia/patologia , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Fosforilase Quinase/genética , Conformação Proteica
13.
Am J Physiol Endocrinol Metab ; 283(1): E29-37, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12067839

RESUMO

Hepatic glucose production is increased in people with type 2 diabetes. Glucose released from storage in liver glycogen by phosphorylase accounts for approximately 50% of the glucose produced after an overnight fast. Therefore, understanding how glycogenolysis in the liver is regulated is of great importance. Toward this goal, we have determined the kinetic characteristics of recombinant human liver glycogen phosphorylase a (HLGPa) (active form) and compared them with those of the purified rat enzyme (RLGPa). The Michaelis-Menten constant (K(m)) of HLGPa for P(i), 5 mM, was about fivefold greater than the K(m) of RLGPa. Two P(i) (substrate) concentrations were used (1 and 5 mM) to cover the physiological range for P(i). Other effectors were added at estimated intracellular concentrations. When added individually, AMP stimulated, whereas ADP, ATP and glucose inhibited, activity. These results were similar to those of the RLGPa. However, glucose inhibition was about twofold more potent with the human enzyme. UDP-glucose, glucose 6-phosphate, and fructose 1-phosphate were only minor inhibitors of both enzymes. We reported previously that when all known effectors were present in combination at physiological concentrations, the net effect was no change in RLGPa activity. However, the same combination reduced HLGPa activity, and the inhibition was glucose dependent. We conclude that a combination of the known effectors of phosphorylase a activity, when present at estimated intracellular concentrations, is inhibitory. Of these effectors, only glucose changes greatly in vivo. Thus it may be the major regulator of HLGPa activity.


Assuntos
Glicogênio Fosforilase Hepática/química , Fígado/enzimologia , Difosfato de Adenosina/farmacologia , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Frutosefosfatos/farmacologia , Glucose/farmacologia , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Humanos , Cinética , Fígado/química , Fosfatos/química , Coelhos , Ratos , Proteínas Recombinantes/química , Especificidade da Espécie , Uridina Difosfato Glucose/farmacologia
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