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1.
J Mol Graph Model ; 61: 231-42, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26364215

RESUMO

Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6]). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC50 values in the range 0.170-0.360µM, with indirubin-3'-acetoxime (1c) the most potent. 7-Bromoindirubin-3'-oxime (13b), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20]) is revealed as a specific inhibitor of PhK (IC50=1.8µM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the 'sum of the modified logarithm of ranks' (SMLR), has been defined which measures performance of a model for both the "early recognition" (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/ß), 6'(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work.


Assuntos
Hipoglicemiantes/química , Indóis/química , Oximas/química , Fosforilase Quinase/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Motivos de Aminoácidos , Sítios de Ligação , Quinase 2 Dependente de Ciclina/química , Quinase 5 Dependente de Ciclina/química , Quinase 3 da Glicogênio Sintase/química , Glicogênio Sintase Quinase 3 beta , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Fosforilase Quinase/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Termodinâmica , Interface Usuário-Computador
2.
Food Chem Toxicol ; 61: 14-27, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23279842

RESUMO

Flavonoids have been discovered as novel inhibitors of glycogen phosphorylase (GP), a target to control hyperglycemia in type 2 diabetes. To elucidate the mechanism of inhibition, we have determined the crystal structure of the GPb-chrysin complex at 1.9 Å resolution. Chrysin is accommodated at the inhibitor site intercalating between the aromatic side chains of Phe285 and Tyr613 through π-stacking interactions. Chrysin binds to GPb approximately 15 times weaker (Ki=19.01 µM) than flavopiridol (Ki=1.24 µM), exclusively at the inhibitor site, and both inhibitors display similar behavior with respect to AMP. To identify the source of flavopiridols' stronger affinity, molecular docking with Glide and postdocking binding free energy calculations using QM/MM-PBSA have been performed and compared. Whereas docking failed to correctly rank inhibitor binding conformations, the QM/MM-PBSA method employing M06-2X/6-31+G to model the π-stacking interactions correctly reproduced the experimental results. Flavopiridols' greater binding affinity is sourced to favorable interactions of the cationic 4-hydroxypiperidin-1-yl substituent with GPb, with desolvation effects limited by the substituent conformation adopted in the crystallographic complex. Further successful predictions using QM/MM-PBSA for the flavonoid quercetagetin (which binds at the allosteric site) leads us to propose the methodology as a useful and inexpensive tool to predict flavonoid binding.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Flavonoides/metabolismo , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/metabolismo , Simulação de Acoplamento Molecular/métodos , Piperidinas/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Cromonas/química , Cromonas/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Flavonas , Flavonoides/química , Flavonoides/farmacologia , Glicogênio Fosforilase/química , Cinética , Modelos Moleculares , Piperidinas/química , Piperidinas/farmacologia , Conformação Proteica , Coelhos , Relação Estrutura-Atividade
3.
Bioorg Med Chem ; 20(5): 1801-16, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22325154

RESUMO

N-(4-Substituted-benzoyl)-N'-(ß-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylated ß-d-glucopyranosyl urea as well as in reactions of O-peracetylated or O-unprotected glucopyranosylamines and acyl-isocyanates. O-deprotections were carried out by base or acid catalysed transesterifications where necessary. Kinetic studies revealed that most of these compounds were low micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). The best inhibitor was the 4-methylbenzoyl compound (K(i)=2.3µM). Crystallographic analyses of complexes of several of the compounds with RMGPb showed that the analogues exploited, together with water molecules, the available space at the ß-pocket subsite and induced a more extended shift of the 280s loop compared to RMGPb in complex with the unsubstituted benzoyl urea. The results suggest the key role of the water molecules in ligand binding and structure-based ligand design. Molecular docking study of selected inhibitors was done to show the ability of the binding affinity prediction. The binding affinity of the highest scored docked poses was calculated and correlated with experimentally measured K(i) values. Results show that correlation is high with the R-squared (R(2)) coefficient over 0.9.


Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Ureia/análogos & derivados , Animais , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Glicogênio Fosforilase/química , Glicogênio Fosforilase/metabolismo , Glicogênio Fosforilase Muscular/antagonistas & inibidores , Glicogênio Fosforilase Muscular/química , Glicogênio Fosforilase Muscular/metabolismo , Modelos Moleculares , Coelhos , Ureia/síntese química , Ureia/química , Ureia/farmacologia
4.
Proteins ; 80(1): 206-20, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22038794

RESUMO

Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP has previously been designated as an adenylate kinase (AK6), but is very atypical as it exhibits unusually broad substrate specificity, structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. Despite its intriguing structure, unique properties and cellular localization, the enzymatic mechanism and biological function of hCINAP have remained poorly characterized. Here, we offer the first high-resolution structure of hCINAP in complex with the substrate ADP (and dADP), the structure of hCINAP with a sulfate ion bound at the AMP binding site, and the structure of the ternary complex hCINAP-Mg(2+) ADP-Pi. Induced fit docking calculations are used to predict the structure of the hCINAP-Mg(2+) ATP-AMP ternary complex. Structural analysis suggested a functional role for His79 in the Walker B motif. Kinetic analysis of mutant hCINAP-H79G indicates that His79 affects both AK and ATPase catalytic efficiency and induces homodimer formation. Finally, we show that in vivo expression of hCINAP-H79G in human cells is toxic and drastically deregulates the number and appearance of CBs in the cell nucleus. Our findings suggest that hCINAP may not simply regulate nucleotide homeostasis, but may have broader functionality, including control of CB assembly and disassembly in the nucleus of human cells.


Assuntos
Adenilato Quinase/química , Proteínas Nucleares/química , Difosfato de Adenosina/química , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Corpos Enovelados/metabolismo , Simulação por Computador , Cristalografia por Raios X , Proteínas de Ligação a DNA , Células HeLa , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Software , Sulfatos/química
5.
Bioorg Med Chem ; 19(17): 5125-36, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21821421

RESUMO

Electrophilic halogenation of C-(2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl) 1,4-dimethoxybenzene (1) afforded regioselectively products halogenated at the para position to the D-glucosyl moiety (8, 9) that were deacetylated to 3 (chloride) and 16 (bromide). For preparing meta regioisomers, 1 was efficiently oxidized with CAN to afford C-(2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl) 1,4-benzoquinone 2 which, in either MeOH or H(2)O-THF containing few equivalents of AcCl, added hydrochloric acid to produce predominantly meta (with respect to the sugar moiety) chlorinated hydroquinone derivatives 5 and 18, this latter being deacetylated to 4. The deacetylated meta (4, 5) or para (3, 16) halohydroquinones were evaluated as inhibitors of glycogen phosphorylase (GP, a molecular target for inhibition of hepatic glycogenolysis under high glucose concentrations) by kinetics and X-ray crystallography. These compounds are competitive inhibitors of GPb with respect to α-D-glucose-1-phosphate. The measured IC(50) values (µM) [169.9±10.0 (3), 95 (4), 39.8±0.3 (5) 136.4±4.9 (16)] showed that the meta halogenated inhibitors (4, 5) are more potent than their para analogs (3, 16). The crystal structures of GPb in complex with these compounds at high resolution (1.97-2.05 Å) revealed that the inhibitors are accommodated at the catalytic site and stabilize the T conformation of the enzyme. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions with protein residues of the different substituents on the aromatic part of the inhibitors.


Assuntos
Inibidores Enzimáticos/síntese química , Glicogênio Fosforilase/antagonistas & inibidores , Halogênios/química , Hidroquinonas/química , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glicogênio Fosforilase/metabolismo , Hidroquinonas/síntese química , Hidroquinonas/metabolismo , Cinética , Conformação Molecular , Ligação Proteica , Estereoisomerismo
6.
Proteins ; 79(3): 703-19, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21287607

RESUMO

With an aim toward glycogenolysis control in Type 2 diabetes, we have investigated via kinetic experiments and computation the potential of indirubin (IC50 > 50 µM), indirubin-3'-oxime (IC50 = 144 nM), KT5720 (K(i) = 18.4 nM) and staurosporine (K(i) = 0.37 nM) as phosphorylase kinase (PhKγtrnc) ATP-binding site inhibitors, with the latter two revealed as potent inhibitors in the low nM range. Because of lack of structural information, we have exploited information from homologous kinase complexes to direct in silico calculations (docking, molecular dynamics, and MMGBSA) to predict the binding characteristics of the four ligands. All inhibitors are predicted to bind in the same active site area as the ATP adenine ring, with binding dominated by hinge region hydrogen bonds to Asp104:O and Met106:O (all four ligands) and also Met106:NH (for the indirubins). The PhKγtrnc-staurosporine complex has the greatest number of receptor-ligand hydrogen bonds, while for the indirubin-3'-oxime and KT5720 complexes there is an important network of interchanging water molecules bridging inhibitor-enzyme contacts. The MM-GBSA results revealed the source of staurosporine's low nM potency to be favorable electrostatic interactions, while KT5720 has strong van der Waals contributions. KT5720 interacts with the greatest number of protein residues either by direct or 1-water bridged hydrogen bond interactions, and the potential for more selective PhK inhibition based on a KT5720 analogue has been established. Including receptor flexibility in Schrödinger induced-fit docking calculations in most cases correctly predicted the binding modes as compared with the molecular dynamics structures; the algorithm was less effective when there were key structural waters bridging receptor-ligand contacts.


Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Carbazóis/química , Fosforilase Quinase/antagonistas & inibidores , Pirróis/química , Estaurosporina/química , Água/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Indóis/química , Cinética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Fosforilase Quinase/química , Homologia de Sequência de Aminoácidos
7.
Bioorg Med Chem ; 18(22): 7911-22, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20947361

RESUMO

Glycogen phosphorylase (GP) is a promising target for the treatment of type 2 diabetes. In the process of structure based drug design for GP, a group of 15 aromatic aldehyde 4-(ß-d-glucopyranosyl)thiosemicarbazones have been synthesized and evaluated as inhibitors of rabbit muscle glycogen phosphorylase b (GPb) by kinetic studies. These compounds are competitive inhibitors of GPb with respect to α-d-glucose-1-phosphate with IC(50) values ranging from 5.7 to 524.3µM. In order to elucidate the structural basis of their inhibition, the crystal structures of these compounds in complex with GPb at 1.95-2.23Å resolution were determined. The complex structures reveal that the inhibitors are accommodated at the catalytic site with the glucopyranosyl moiety at approximately the same position as α-d-glucose and stabilize the T conformation of the 280s loop. The thiosemicarbazone part of the studied glucosyl thiosemicarbazones possess a moiety derived from substituted benzaldehydes with NO(2), F, Cl, Br, OH, OMe, CF(3), or Me at the ortho-, meta- or para-position of the aromatic ring as well as a moiety derived from 4-pyridinecarboxaldehyde. These fit tightly into the ß-pocket, a side channel from the catalytic site with no access to the bulk solvent. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions of the aldehyde-derived moiety with protein residues in the ß-pocket. In addition, 14 out of the 15 studied inhibitors were found bound at the new allosteric site of the enzyme.


Assuntos
Inibidores Enzimáticos/química , Glucose/química , Glicogênio Fosforilase Muscular/antagonistas & inibidores , Tiossemicarbazonas/química , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glucofosfatos/química , Glicogênio Fosforilase Muscular/metabolismo , Halogênios/química , Cinética , Conformação Molecular , Ligação Proteica , Piridinas/química , Coelhos , Tiossemicarbazonas/síntese química , Tiossemicarbazonas/farmacologia
8.
Bioorg Med Chem ; 17(20): 7368-80, 2009 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-19781947

RESUMO

A series of glucopyranosylidene-spiro-isoxazolines was prepared through regio- and stereoselective [3+2]-cycloaddition between the methylene acetylated exo-glucal and aromatic nitrile oxides. The deprotected cycloadducts were evaluated as inhibitors of muscle glycogen phosphorylase b. The carbohydrate-based family of five inhibitors displays K(i) values ranging from 0.63 to 92.5 microM. The X-ray structures of the enzyme-ligand complexes show that the inhibitors bind preferentially at the catalytic site of the enzyme retaining the less active T-state conformation. Docking calculations with GLIDE in extra-precision (XP) mode yielded excellent agreement with experiment, as judged by comparison of the predicted binding modes of the five ligands with the crystallographic conformations and the good correlation between the docking scores and the experimental free binding energies. Use of docking constraints on the well-defined positions of the glucopyranose moiety in the catalytic site and redocking of GLIDE-XP poses using electrostatic potential fit-determined ligand partial charges in quantum polarized ligand docking (QPLD) produced the best results in this regard.


Assuntos
Inibidores Enzimáticos/farmacologia , Glucose/química , Glicogênio Fosforilase/antagonistas & inibidores , Oxazóis/farmacologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Glicogênio Fosforilase/metabolismo , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Oxazóis/química , Espectrometria de Massas por Ionização por Electrospray
9.
Structure ; 17(1): 117-27, 2009 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-19141288

RESUMO

Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a. We present a 9.9 angstroms resolution structure of PhK heterotetramer (alphabetagammadelta)4 determined by cryo-electron microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic gamma subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 angstroms resolution, which shows the location of the substrate near the kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 angstroms resolution was consistent with a proteolysed activated form of PhK that had lost the alpha subunits and possibly the gamma subunits.


Assuntos
Glicogênio Fosforilase/metabolismo , Fosforilase Quinase/química , Domínio Catalítico , Cromatografia em Gel , Microscopia Crioeletrônica , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Fosforilase Quinase/metabolismo , Conformação Proteica , Especificidade por Substrato
10.
J Med Chem ; 52(4): 932-42, 2009 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-19173562

RESUMO

Six 5'-deoxy-5'-morpholine, piperidine, and pyrrolidine of pyrimidine nucleosides have been synthesized and characterized. Their inhibitory action to ribonuclease A has been studied by biochemical analysis and X-ray crystallography. These compounds are moderate inhibitors of RNase A with mid-to-upper micromolar inhibition constants (K(i)). The high resolution X-ray crystal structures of the RNase A-inhibitor complexes have shown that all inhibitors bind at the enzyme catalytic cleft with the pyrimidine nucleobase at the B(1)R(2) subsites while the 5' group binds away from the main subsite P(1), where P-O(5') bond cleavage occurs, toward the solvent close to subsite P(0). Structure-activity relationship analysis has demonstrated that the compounds with the larger group in the 5' position are more potent. Comparative structural analysis of these RNase A complexes with other similar RNase A-ligand complexes provides a structural explanation of their potency and suggests ways to improve their efficiency and selectivity. These inhibitors can be the starting point for the development of compounds that can be used as pharmaceuticals against pathologies associated with RNase A homologues such as human angiogenin, which is implicated in tumor induced neovascularization.


Assuntos
Nucleosídeos de Pirimidina/síntese química , Ribonuclease Pancreático/antagonistas & inibidores , Domínio Catalítico , Cristalografia por Raios X , Humanos , Morfolinas , Neovascularização Patológica/tratamento farmacológico , Piperidinas , Ligação Proteica , Conformação Proteica , Nucleosídeos de Pirimidina/química , Nucleosídeos de Pirimidina/farmacologia , Pirrolidinas , Relação Estrutura-Atividade
11.
J Med Chem ; 51(12): 3540-54, 2008 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-18517260

RESUMO

Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this study, were biologically evaluated as inhibitors of rabbit muscle glycogen phosphorylase a (GPa). From SAR studies, the presence of a sugar moiety in triterpene saponins resulted in a markedly decreased activity ( 7, 18- 20) or no activity ( 21, 22). These saponins, however, might find their value as potential natural prodrugs which are much more water-soluble than their corresponding aglycones. To elucidate the mechanism of GP inhibition, we have determined the crystal structures of the GPb-asiatic acid and GPb-maslinic acid complexes. The X-ray analysis indicates that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Pentacyclic triterpenes represent a promising class of multiple-target antidiabetic agents that exert hypoglycemic effects, at least in part, through GP inhibition.


Assuntos
Glicogênio Fosforilase/antagonistas & inibidores , Hipoglicemiantes/síntese química , Modelos Moleculares , Triterpenos/síntese química , Monofosfato de Adenosina/química , Sítio Alostérico , Animais , Sítios de Ligação , Cristalografia por Raios X , Glicogênio Fosforilase/química , Hipoglicemiantes/química , Cinética , Músculos/enzimologia , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/síntese química , Ácido Oleanólico/química , Triterpenos Pentacíclicos , Ligação Proteica , Conformação Proteica , Coelhos , Estereoisomerismo , Relação Estrutura-Atividade , Triterpenos/química
12.
Curr Opin Investig Drugs ; 9(4): 379-95, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18393105

RESUMO

The regulation of glycogen metabolism is a major therapeutic strategy for blood glucose control in type 2 diabetes. Because glycogen phosphorylase catalyzes the first step in the phosphorolysis of glycogen, it has become a potential key target for controlling hyperglycemia in this disease. This review focuses on advances in new, mostly synthetic, molecules that inhibit glycogen phosphorylase, and describes progress in our understanding of the mechanism of action of these inhibitors gained through X-ray crystallographic studies.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Glicogênio Fosforilase/química , Hipoglicemiantes/química , Tecnologia Farmacêutica/métodos , Regulação Alostérica , Sítio Alostérico , Animais , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glicogênio/metabolismo , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/metabolismo , Humanos , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
13.
Chembiochem ; 9(2): 253-60, 2008 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-18188862

RESUMO

Several members of a new family of seven-membered azasugars, which can be seen as 1-azasugar ring homologues, have been obtained by simple chemical transformations starting from a sugar-derived azidolactol. Unlike their piperidine counterparts, these molecules are chemically stable when they possess a hydroxy group at the pseudo-C-2 position. Biological assays with a range of carbohydrate-processing enzymes have revealed interesting potential for these compounds. A trihydroxymethyl-substituted azepane displayed strong competitive inhibition on almond beta-glucosidase (K(i)=2.5 microM) while a trihydroxylated carboxylic acid derivative proved to be a potent and selective L-fucosidase inhibitor (K(i)=41 nM). N-Butylation of these seven-membered 1-azasugars generated derivatives with some activity towards the Gaucher's disease-related glucosylceramide transferase (IC(50) 75 microM) that did not interact significantly with digestive glucosidases.


Assuntos
Compostos Aza/síntese química , Azepinas/síntese química , Carboidratos/síntese química , Inibidores Enzimáticos/síntese química , Glucosilceramidas/antagonistas & inibidores , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosiltransferases/antagonistas & inibidores , Compostos Aza/farmacologia , Azepinas/farmacologia , Bioensaio , Carboidratos/farmacologia , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/patologia
14.
Proteins ; 71(3): 1307-23, 2008 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-18041758

RESUMO

4-Phenyl-N-(beta-D-glucopyranosyl)-1H-1,2,3-triazole-1-acetamide (glucosyltriazolylacetamide) has been studied in kinetic and crystallographic experiments with glycogen phosphorylase b (GPb), in an effort to utilize its potential as a lead for the design of potent antihyperglycaemic agents. Docking and molecular dynamics (MD) calculations have been used to monitor more closely the binding modes in operation and compare the results with experiment. Kinetic experiments in the direction of glycogen synthesis showed that glucosyltriazolylacetamide is a better inhibitor (K(i) = 0.18 mM) than the parent compound alpha-D-glucose (K(i) = 1.7 mM) or beta-D-glucose (K(i) = 7.4 mM) but less potent inhibitor than the lead compound N-acetyl-beta-D-glucopyranosylamine (K(i) = 32 microM). To elucidate the molecular basis underlying the inhibition of the newly identified compound, we determined the structure of GPb in complex with glucosyltriazolylacetamide at 100 K to 1.88 A resolution, and the structure of the compound in the free form. Glucosyltriazolylacetamide is accommodated in the catalytic site of the enzyme and the glucopyranose interacts in a manner similar to that observed in the GPb-alpha-D-glucose complex, while the substituent group in the beta-position of the C1 atom makes additional hydrogen bonding and van der Waals interactions to the protein. A bifurcated donor type hydrogen bonding involving O3H, N3, and N4 is seen as an important structural motif strengthening the binding of glucosyltriazolylacetamide with GP which necessitated change in the torsion about C8-N2 bond by about 62 degrees going from its free to the complex form with GPb. On binding to GP, glucosyltriazolylacetamide induces significant conformational changes in the vicinity of this site. Specifically, the 280s loop (residues 282-288) shifts 0.7 to 3.1 A (CA atoms) to accommodate glucosyltriazolylacetamide. These conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding compared with the lead compound. In the molecular modeling calculations, the GOLD docking runs with and without the crystallographic ordered cavity waters using the GoldScore scoring function, and without cavity waters using the ChemScore scoring function successfully reproduced the crystallographic binding conformation. However, the GLIDE docking calculations both with (GLIDE XP) and without (GLIDE SP and XP) the cavity water molecules were, impressively, further able to accurately reproduce the finer details of the GPb-glucosyltriazolylacetamide complex structure. The importance of cavity waters in flexible receptor MD calculations compared to "rigid" (docking) is analyzed and highlighted, while in the MD itself very little conformational flexibility of the glucosyltriazolylacetamide ligand was observed over the time scale of the simulations.


Assuntos
Azidas/química , Biologia Computacional , Cristalografia por Raios X , Glucosamina/análogos & derivados , Glucose/análogos & derivados , Glucose/química , Glicogênio Fosforilase/antagonistas & inibidores , Ureia/análogos & derivados , Animais , Sítios de Ligação , Inibidores Enzimáticos/química , Glucosamina/química , Glicogênio Fosforilase/química , Glicogênio Fosforilase/metabolismo , Ligação Proteica , Coelhos , Ureia/química
15.
Protein Sci ; 16(8): 1773-82, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17600143

RESUMO

FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b-FR258900 complex and refined it to 2.2 A resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45' (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy-phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site.


Assuntos
Cinamatos/química , Glutaratos/química , Glicogênio Fosforilase Muscular/química , Hipoglicemiantes/química , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Sítio Alostérico , Animais , Benzamidas/química , Benzamidas/metabolismo , Sítios de Ligação , Cinamatos/metabolismo , Glutaratos/metabolismo , Glicogênio Fosforilase Muscular/metabolismo , Humanos , Hipoglicemiantes/metabolismo , Ligantes , Modelos Moleculares , Compostos de Fenilureia/química , Ácidos Ftálicos/química , Ácidos Ftálicos/metabolismo , Conformação Proteica , Coelhos , Ratos
16.
J Mol Biol ; 368(4): 1145-61, 2007 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-17391699

RESUMO

The crystal structure of a novel fungal lectin from Sclerotium rolfsii (SRL) in its free form and in complex with N-acetyl-d-galactosamine (GalNAc) and N-acetyl- d -glucosamine (GlcNAc) has been determined at 1.1 A, 2.0 A, and 1.7 A resolution, respectively. The protein structure is composed of two beta-sheets, which consist of four and six beta-strands, connected by two alpha-helices. Sequence and structural comparisons reveal that SRL is the third member of a newly identified family of fungal lectins, which includes lectins from Agaricus bisporus and Xerocomus chrysenteron that share a high degree of structural similarity and carbohydrate specificity. The data for the free SRL are the highest resolution data for any protein of this family. The crystal structures of the SRL in complex with two carbohydrates, GalNAc and GlcNAc, which differ only in the configuration of a single epimeric hydroxyl group, provide the structural basis for its carbohydrate specificity. SRL has two distinct carbohydrate-binding sites, a primary and a secondary. GalNAc binds at the primary site, whereas GlcNAc binds only at the secondary site. Thus, SRL has the ability to recognize and probably bind at the same time two different carbohydrate structures. Structural comparison to Agaricus bisporus lectin-carbohydrate complexes reveals that the primary site is also able to bind the Thomsen-Friedenreich antigen (Galbeta1-->3GalNAc-alpha- glycan structures) whereas the secondary site cannot. The features of the molecular recognition at the two sites are described in detail.


Assuntos
Acetilgalactosamina/metabolismo , Acetilglucosamina/metabolismo , Proteínas Fúngicas/química , Lectinas/química , Modelos Moleculares , Polyporales/química , Sequência de Aminoácidos , Proteínas Fúngicas/metabolismo , Lectinas/metabolismo , Dados de Sequência Molecular , Conformação Proteica
17.
Biophys J ; 92(5): 1659-72, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17142283

RESUMO

The proteins of the pancreatic ribonuclease A (RNase A) family catalyze the cleavage of the RNA polymer chain. The development of RNase inhibitors is of significant interest, as some of these compounds may have a therapeutic effect in pathological conditions associated with these proteins. The most potent low molecular weight inhibitor of RNase reported to date is the compound 5'-phospho-2'-deoxyuridine-3-pyrophosphate (P-->5)-adenosine-3-phosphate (pdUppA-3'-p). The 3',5'-pyrophosphate group of this compound increases its affinity and introduces structural features which seem to be unique in pyrophosphate-containing ligands bound to RNase A, such as the adoption of a syn conformation by the adenosine base at RNase subsite B(2) and the placement of the 5'-beta-phosphate of the adenylate (instead of the alpha-phosphate) at subsite P(1) where the phosphodiester bond cleavage occurs. In this work, we study by multi-ns molecular dynamics simulations the structural properties of RNase A complexes with the ligand pdUppA-3'-p and the related weaker inhibitor dUppA, which lacks the 3' and 5' terminal phosphate groups of pdUppA-3'-p. The simulations show that the adenylate 5'-beta-phosphate binding position and the adenosine syn orientation constitute robust structural features in both complexes, stabilized by persistent interactions with specific active-site residues of subsites P(1) and B(2). The simulation structures are used in conjunction with a continuum-electrostatics (Poisson-Boltzmann) model, to evaluate the relative binding affinity of the two complexes. The computed relative affinity of pdUppA-3'-p varies between -7.9 kcal/mol and -2.8 kcal/mol for a range of protein/ligand dielectric constants (epsilon(p)) 2-20, in good agreement with the experimental value (-3.6 kcal/mol); the agreement becomes exact with epsilon(p) = 8. The success of the continuum-electrostatics model suggests that the differences in affinity of the two ligands originate mainly from electrostatic interactions. A residue decomposition of the electrostatic free energies shows that the terminal phosphate groups of pdUppA-3'-p make increased interactions with residues Lys(7) and Lys(66) of the more remote sites P(2) and P(0), and His(119) of site P(1).


Assuntos
Monofosfato de Adenosina/análogos & derivados , Nucleotídeos de Desoxiuracil/química , Ribonuclease Pancreático/química , Monofosfato de Adenosina/química , Sítios de Ligação , Simulação por Computador , Ligantes , Conformação Proteica , Ribonuclease Pancreático/antagonistas & inibidores
18.
Bioorg Med Chem Lett ; 17(2): 394-9, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17095214

RESUMO

A series of substituted 3,4-dihydro-2-quinolone glycogen phosphorylase inhibitors, which have potential as antidiabetic agents, is described. Initial members of the series showed good enzyme inhibitory potency but poor physical properties. Optimisation of the 1-substituent led to 2,3-dihydroxypropyl compounds which showed good in vitro potency and improved physical properties, together with good DMPK profiles and acute in vivo efficacy in a rat model. X-ray crystallographic data are presented, showing an unexpected variety of binding orientations at the dimer interface site.


Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Quinolinas/síntese química , Quinolinas/farmacologia , Animais , Disponibilidade Biológica , Proteínas Sanguíneas/metabolismo , Células CACO-2 , Fenômenos Químicos , Físico-Química , Cristalografia por Raios X , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Indicadores e Reagentes , Fígado/enzimologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Músculo Esquelético/enzimologia , Coelhos , Ratos
19.
Bioorg Med Chem Lett ; 16(21): 5567-71, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16945526

RESUMO

Two series of novel thienopyrrole inhibitors of recombinant human liver glycogen phosphorylase a (GPa) which are effective in reducing glucose output from rat hepatocytes are described. Representative compounds have been shown to bind at the dimer interface site of the rabbit muscle enzyme by X-ray crystallography.


Assuntos
Glicogênio Fosforilase/antagonistas & inibidores , Pirróis/farmacologia , Animais , Cristalografia por Raios X , Humanos , Pirróis/síntese química , Pirróis/química , Coelhos , Ratos , Relação Estrutura-Atividade
20.
J Med Chem ; 49(19): 5687-701, 2006 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-16970395

RESUMO

Iminosugars DAB (5), isofagomine (9), and several N-substituted derivatives have been identified as potent inhibitors of liver glycogen phosphorylase a (IC(50) = 0.4-1.2 microM) and of basal and glucagon-stimulated glycogenolysis (IC(50) = 1-3 microM). The X-ray structures of 5, 9, and its N-3-phenylpropyl analogue 8 in complex with rabbit muscle glycogen phosphorylase (GPb) shows that iminosugars bind tightly at the catalytic site in the presence of the substrate phosphate and induce conformational changes that characterize the R-state conformation of the enzyme. Charged nitrogen N1 is within hydrogen-bonding distance with the carbonyl oxygen of His377 (5) and in ionic contact with the substrate phosphate oxygen (8 and 9). Our findings suggest that the inhibitors function as oxocarbenium ion transition-state analogues. The conformational change to the R state provides an explanation for previous findings that 5, unlike inhibitors that favor the T state, promotes phosphorylation of GPb in hepatocytes with sequential inactivation of glycogen synthase.


Assuntos
Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/química , Imino Açúcares/síntese química , Animais , Domínio Catalítico , Cristalografia por Raios X , Glucofosfatos/química , Glicogenólise , Imino Açúcares/química , Cinética , Fígado/enzimologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Coelhos , Estereoisomerismo , Relação Estrutura-Atividade
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