RESUMO
Psoriasis is a skin inflammatory disorder that affects 3% of the human population. Although several therapies based on the neutralization of proinflammatory cytokines have been used with relative success, additional treatments are required. The in silico analysis of gene expression data of psoriasis lesional skin and an analysis of vitamin B6 metabolites in the sera of psoriasis patients point to altered vitamin B6 metabolism at both local and systemic levels. Functional studies showed that vitamin B6 vitamers reduced skin neutrophil infiltration, oxidative stress and Nfkb activity in two zebrafish models of skin inflammation. Strikingly, inhibition of glycogen phosphorylase L (Pygl) and glucose-6-phosphate dehydrogenase (G6pd), two vitamin B6-regulated enzymes, alleviated oxidative-stress induced inflammation in zebrafish skin inflammation models. Despite the central role of G6pd in antioxidant defenses, the results of the study demonstrate that glycogen stores and G6pd fuel NADPH oxidase to promote skin inflammation, revealing novel targets for the treatment of skin inflammatory disorders.
Assuntos
Glucosefosfato Desidrogenase/metabolismo , Glicogênio Fosforilase Hepática/metabolismo , Psoríase/imunologia , Vitamina B 6/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Biópsia , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/genética , Glicogênio/metabolismo , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Glicogênio Fosforilase Hepática/genética , Células HaCaT , Humanos , Microscopia Intravital , NADPH Oxidases/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Psoríase/sangue , Psoríase/tratamento farmacológico , Psoríase/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Pele/diagnóstico por imagem , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Vitamina B 6/sangue , Peixe-ZebraRESUMO
C-Glucopyranosyl imidazoles, thiazoles, and an N-glucopyranosyl tetrazole were assessed in vitro and ex vivo for their inhibitory efficiency against isoforms of glycogen phosphorylase (GP; a validated pharmacological target for the development of anti-hyperglycaemic agents). Imidazoles proved to be more potent inhibitors than the corresponding thiazoles or the tetrazole. The most potent derivative has a 2-naphthyl substituent, a Ki value of 3.2 µM for hepatic glycogen phosphorylase, displaying also 60% inhibition of GP activity in HepG2 cells, compared to control vehicle treated cells, at 100 µM. X-Ray crystallography studies of the protein - inhibitor complexes revealed the importance of the architecture of inhibitor associated hydrogen bonds or sulfur σ-hole bond interactions to Asn284 OD1, offering new insights to structure-based design efforts. Moreover, while the 2-glucopyranosyl-tetrazole seems to bind differently from the corresponding 1,2,3-triazole compound, the two inhibitors are equipotent.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Imidazóis/farmacologia , Tetrazóis/farmacologia , Tiazóis/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glicogênio Fosforilase Hepática/metabolismo , Células Hep G2 , Humanos , Hidrogênio/química , Imidazóis/síntese química , Imidazóis/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Enxofre/química , Tetrazóis/síntese química , Tetrazóis/química , Tiazóis/síntese química , Tiazóis/químicaRESUMO
Glycogen phosphorylase (GP) is a validated target for the development of new type 2 diabetes treatments. Exploiting the Zinc docking database, we report the in silico screening of 1888 N-acyl-ß-d-glucopyranosylamines putative GP inhibitors differing only in their R groups. CombiGlide and GOLD docking programs with different scoring functions were employed with the best performing methods combined in a 'consensus scoring' approach to ranking of ligand binding affinities for the active site. Six selected candidates from the screening were then synthesized and their inhibitory potency was assessed both in vitro and ex vivo. Their inhibition constants' values, in vitro, ranged from 5 to 377µM while two of them were effective at causing inactivation of GP in rat hepatocytes at low µM concentrations. The crystal structures of GP in complex with the inhibitors were defined and provided the structural basis for their inhibitory potency and data for further structure based design of more potent inhibitors.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Glucosamina/análogos & derivados , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Glucosamina/síntese química , Glucosamina/química , Glucosamina/farmacologia , Glicogênio Fosforilase Hepática/metabolismo , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The effects of a glycogen phosphorylase inhibitor (GPI) and metformin (MT) on hepatic glucose fluxes (µmol · kg(-1) · min(-1)) in the presence of basal and 4-fold basal levels of plasma glucagon were investigated in 18-h fasted conscious dogs. Compared with the vehicle treatment, GPI infusion suppressed net hepatic glucose output (NHGO) completely (-3.8 ± 1.3 versus 9.9 ± 2.8) despite increased glucose 6-phosphate (G-6-P) neogenesis from gluconeogenic precursors (8.1 ± 1.1 versus 5.5 ± 1.1). MT infusion did not alter those parameters. In response to a 4-fold rise in plasma glucagon levels, in the vehicle group, plasma glucose levels were increased 2-fold, and NHGO was increased (43.9 ± 5.7 at 10 min and 22.7 ± 3.4 at steady state) without altering G-6-P neogenesis (3.7 ± 1.5 and 5.5 ± 0.5, respectively). In the GPI group, there was no increase in NHGO due to decreased glucose-6-phosphatase flux associated with reduced G-6-P concentration. A lower G-6-P concentration was the result of increased net glycogenesis without altering G-6-P neogenesis. In the MT group, the increment in NHGO (22.2 ± 4.4 at 10 min and 12.1 ± 3.6 at steady state) was approximately half of that of the vehicle group. The lesser NHGO was associated with reduced glucose-6-phosphatase flux but a rise in G-6-P concentration and only a small incorporation of plasma glucose into glycogen. In conclusion, the inhibition of glycogen phosphorylase a activity decreases basal and glucagon-induced NHGO via redirecting glucose 6-phosphate flux from glucose toward glycogen, and MT decreases glucagon-induced NHGO by inhibiting glucose-6-phosphatase flux and thereby reducing glycogen breakdown.
Assuntos
Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Glicogênio Hepático/metabolismo , Fígado/efeitos dos fármacos , Metformina/farmacologia , Animais , Glicemia/metabolismo , Cães , Jejum , Ácidos Graxos não Esterificados/sangue , Ácidos Graxos não Esterificados/metabolismo , Feminino , Glucagon/sangue , Glucagon/metabolismo , Glucagon/farmacologia , Gluconeogênese/efeitos dos fármacos , Gluconeogênese/fisiologia , Glucose-6-Fosfatase/efeitos dos fármacos , Glucose-6-Fosfatase/fisiologia , Glicerol/sangue , Glicerol/metabolismo , Glicogênio Fosforilase Hepática/metabolismo , Hematócrito , Indóis/farmacologia , Insulina/sangue , Insulina/metabolismo , Ácido Láctico/sangue , Ácido Láctico/metabolismo , Fígado/metabolismo , Masculino , Fenilbutiratos/farmacologiaRESUMO
A series of carboxylic acid glycogen phosphorylase inhibitors, which have potential as oral antidiabetic agents, is described. Defining and applying simple physicochemical design criteria was used to assess the opportunity and to focus synthetic efforts on compounds with the greatest probability of success. The study led to compound 17, which exhibits a good balance of properties including potent inhibition of recombinant human liver glycogen phosphorylase in vitro, a good DMPK profile including excellent bioavailability and low clearance and good in vivo activity in a glucagon challenge model of diabetes in Zucker rats.
Assuntos
Ácidos Carboxílicos/farmacologia , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Hipoglicemiantes/química , Indanos/farmacologia , Animais , Disponibilidade Biológica , Ácidos Carboxílicos/química , Ácidos Carboxílicos/uso terapêutico , Descoberta de Drogas , Humanos , Hipoglicemiantes/farmacologia , Indanos/química , Indanos/uso terapêutico , Ratos , Ratos ZuckerRESUMO
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/químicaRESUMO
During our research using a high-throughput screening system for discovery of a new class of human liver glycogen phosphorylase a (hLGPa) inhibitors, a series of 3-(3,4-dichlorophenyl)acrylamide derivatives were synthesized, and their inhibitory activities toward hLGPa were evaluated. Among the derivatives, (2E,2'E)-N,N'-pentane-1,5-diylbis[3-(3,4-dichlorophenyl)acrylamide] (6c) inhibited hLGPa with an IC(50) value of 0.023 microM. An X-ray crystallographic study of the enzyme-6c complex showed that the inhibitor is bound at the dimer interface site, where the 3,4-dichlorophenyl moiety interacts hydrophobically with the enzyme.
Assuntos
Acrilamidas/farmacologia , Diclorofeno/farmacologia , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Acrilamidas/síntese química , Sítios de Ligação , Cristalografia por Raios X , Diclorofeno/síntese química , Inibidores Enzimáticos/síntese química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Hipoglicemiantes/síntese química , Solventes/química , Relação Estrutura-AtividadeRESUMO
The synthesis of carbohydrate-based glycogen phosphorylase inhibitors is attractive for potential applications in the treatment of type 2 diabetes. A titanium-mediated synthesis led to a benzoylated C-glucosylated cyclopropylamine intermediate, which underwent a benzoyl migration to afford the corresponding 2-hydroxy-C-glycoside. X-ray crystallographic studies revealed a unit cell composed of four molecules as pairs of dimers connected through two hydrogen bonds. The deprotection of the benzoate esters under Zemplén conditions afforded a glycogen phosphorylase inhibitor candidate displaying weak inhibition toward glycogen phosphorylase (16% at 2.5mM).
Assuntos
Benzamidas/síntese química , Benzamidas/farmacologia , Glicogênio Fosforilase Muscular/antagonistas & inibidores , Glicogênio/metabolismo , Animais , Cristalografia por Raios X , Dimerização , Avaliação Pré-Clínica de Medicamentos , Glucosídeos/síntese química , Glucosídeos/farmacologia , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Glicogênio Fosforilase Hepática/metabolismo , Glicogênio Fosforilase Muscular/metabolismo , Glicosilação , CoelhosRESUMO
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/farmacologiaRESUMO
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 , EstereoisomerismoRESUMO
Rabbit muscle glycogen phosphorylase-a (GPa) reduces arsenate (As(V)) to the more toxic arsenite (As(III)) in a glutathione (GSH)-dependent fashion. To determine whether reduction of As(V) by GPa is countered by compounds known to inhibit GP-catalyzed glycogenolysis, the effects of thiol reagents, endogenous compounds (glucose, ATP, ADP) as well as nonspecific glycogen phosphorylase inhibitors (GPIs; caffeine, quercetin, flavopiridol [FP]), and specific GPIs (1,4-dideoxy-1,4-imino-D-arabinitol [DAB], BAY U6751, CP320626) were tested on reduction of As(V) by rabbit muscle GPa in the presence of glycogen (substrate), AMP (activator), and GSH, and the As(III) formed from As(V) was quantified by high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry. The As(V)-reducing activity of GPa was moderately sensitive to thiol reagents. Glucose above 5mM and ADP or ATP at physiological levels diminished GPa-catalyzed As(V) reduction. All GPIs inhibited As(V) reduction by GPa in a concentration-dependent fashion; however, their effects were differentially affected by glucose (10mM) or AMP (200microM instead of 25microM), known modulators of the action of some GPIs on the GP-catalyzed glycogenolysis. Inhibition of As(V) reduction by DAB and quercetin was not influenced by glucose or AMP. Glucose that potentiates the inhibitory effects of caffeine, BAY U6751, and CP320626 on the glycogenolytic activity of GPa also enhanced the inhibitory effects of these GPIs on GPa-catalyzed As(V) reduction. AMP at high concentration alleviated the inhibition by BAY U6751 and CP320626 (whose antagonistic effect on GP-catalyzed glycogen breakdown is also AMP sensitive), whereas the inhibition in As(V) reduction by FP or caffeine was little affected by AMP. Thus, GPIs inhibit both the glycogenolytic and As(V)-reducing activities of GP, supporting that the latter is coupled to glycogenolysis. It was also shown that a GPa-rich extract of rat liver contained GSH-dependent As(V)-reducing activity that was inhibited by specific GPIs, suggesting that the liver-type GPa can also catalyze reduction of As(V).
Assuntos
Arseniatos/metabolismo , Arsenitos/metabolismo , Inibidores Enzimáticos/farmacologia , Glutationa/metabolismo , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Glicogênio Fosforilase Muscular/antagonistas & inibidores , Glicogenólise/efeitos dos fármacos , Xenobióticos/farmacologia , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Amidas/farmacologia , Animais , Arabinose/farmacologia , Cafeína/farmacologia , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Glucose/metabolismo , Glucose-6-Fosfato/metabolismo , Glicogênio/metabolismo , Glicogênio Fosforilase Hepática/metabolismo , Glicogênio Fosforilase Muscular/metabolismo , Imino Furanoses/farmacologia , Indóis/farmacologia , Masculino , Oxirredução , Quercetina/farmacologia , Ácidos Quinolínicos/farmacologia , Coelhos , Ratos , Ratos Wistar , Espectrometria de Fluorescência , Espectrofotometria Atômica , Álcoois Açúcares/farmacologia , Reagentes de Sulfidrila/farmacologiaRESUMO
Glycogen phosphorylase inhibition represents a promising strategy to suppress inappropriate hepatic glucose output, while muscle glycogen is a major source of fuel during contraction. Glycogen phosphorylase inhibitors (GPi) currently being investigated for the treatment of type 2 diabetes do not demonstrate hepatic versus muscle glycogen phosphorylase isoform selectivity and may therefore impair patient aerobic exercise capabilities. Skeletal muscle energy metabolism and function are not impaired by GPi during high-intensity contraction in rat skeletal muscle; however, it is unknown whether glycogen phosphorylase inhibitors would impair function during prolonged lower-intensity contraction. Utilizing a novel red cell-perfused rodent gastrocnemius-plantaris-soleus system, muscle was pretreated for 60 min with either 3 micromol/l free drug GPi (n=8) or vehicle control (n=7). During 60 min of aerobic contraction, GPi treatment resulted in approximately 35% greater fatigue. Muscle glycogen phosphorylase a form (P<0.01) and maximal activity (P<0.01) were reduced in the GPi group, and postcontraction glycogen (121.8 +/- 16.1 vs. 168.3 +/- 8.5 mmol/kg dry muscle, P<0.05) was greater. Furthermore, lower muscle lactate efflux and glucose uptake (P<0.01), yet higher muscle Vo(2), support the conclusion that carbohydrate utilization was impaired during contraction. Our data provide new confirmation that muscle glycogen plays an essential role during submaximal contraction. Given the critical role of exercise prescription in the treatment of type 2 diabetes, it will be important to monitor endurance capacity during the clinical evaluation of nonselective GPi. Alternatively, greater effort should be devoted toward the discovery of hepatic-selective GPi, hepatic-specific drug delivery strategies, and/or alternative strategies for controlling excess hepatic glucose production in type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Glucose/metabolismo , Glicogênio/metabolismo , Glicogênio Fosforilase Hepática/antagonistas & inibidores , Glicogênio Fosforilase Muscular/antagonistas & inibidores , Ácido Láctico/metabolismo , Glicogênio Hepático/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Ratos , Ratos WistarRESUMO
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 ProteicaRESUMO
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/químicaRESUMO
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/farmacologiaRESUMO
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 , TermodinâmicaRESUMO
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.