ABSTRACT
Recently studied N-(ß-d-glucopyranosyl)-3-aryl-1,2,4-triazole-5-carboxamides have proven to be low micromolar inhibitors of glycogen phosphorylase (GP), a validated target for the treatment of type 2 diabetes mellitus. Since in other settings, the bioisosteric replacement of the 1,2,4-triazole moiety with imidazole resulted in significantly more efficient GP inhibitors, in silico calculations using Glide molecular docking along with unbound state DFT calculations were performed on N-(ß-d-glucopyranosyl)-arylimidazole-carboxamides, revealing their potential for strong GP inhibition. The syntheses of the target compounds involved the formation of an amide bond between per-O-acetylated ß-d-glucopyranosylamine and the corresponding arylimidazole-carboxylic acids. Kinetics experiments on rabbit muscle GPb revealed low micromolar inhibitors, with the best inhibition constants (Kis) of ~3-4 µM obtained for 1- and 2-naphthyl-substituted N-(ß-d-glucopyranosyl)-imidazolecarboxamides, 2b-c. The predicted protein-ligand interactions responsible for the observed potencies are discussed and will facilitate the structure-based design of other inhibitors targeting this important therapeutic target. Meanwhile, the importance of the careful consideration of ligand tautomeric states in binding calculations is highlighted, with the usefulness of DFT calculations in this regard proposed.
Subject(s)
Enzyme Inhibitors , Glycogen Phosphorylase , Imidazoles , Molecular Docking Simulation , Kinetics , Rabbits , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemical synthesis , Glycogen Phosphorylase/antagonists & inhibitors , Glycogen Phosphorylase/metabolism , Glycogen Phosphorylase/chemistry , Imidazoles/chemistry , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Computer Simulation , Structure-Activity Relationship , Triazoles/chemistry , Triazoles/pharmacology , Triazoles/chemical synthesisABSTRACT
The accumulation of rabbit muscle glycogen phosphorylase b (RMGPb) in electrostatic complexes with the cationic polyelectrolyte poly 2-(dimethylamino) ethyl methacrylate in its quenched form (QPDMAEMA) was studied in two buffer solutions. In the N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, large complexes of RMGPb-QPDMAEMA were formed which adopted smaller sizes as QPDMAEMA concentration increased. However, in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer, the hydrodynamic radius of the formed complexes gradually increased as the polymer concentration increased. Zeta potential measurements (ζp) showed that RMGPb significantly changed the ζp of the QPDMAEMA aggregates. Fluorescence studies showed that the interaction between RMGPb and QPDMAEAMA was enhanced as polymer concentration increased. Specifically, 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence indicated that in the BES buffer the aggregates became denser as more QPDMAEMA was added, while in the HEPES buffer the density of the formed structures decreased. RMGPb's secondary structure was examined by Attenuated Total Reflection - Fourier Transform Infrared (ATR-FTIR) and Circular Dichroism (CD) showing that QPDMAEMA interaction with RMGPb does not induce any changes to the secondary structure of the enzyme. These observations suggest that cationic polyelectrolytes may be utilized for the formulation of RMGPb in multifunctional nanostructures and be further exploited in innovative biotechnology applications and bioinspired materials development.
Subject(s)
Glycogen Phosphorylase , Polymers , Animals , Cations , Glycogen Phosphorylase/chemistry , HEPES , Polyelectrolytes , Polymers/chemistry , RabbitsABSTRACT
The crystal structures of free T-state and R-state glycogen phosphorylase (GP) and of R-state GP in complex with the allosteric activators IMP and AMP are reported at improved resolution. GP is a validated pharmaceutical target for the development of antihyperglycaemic agents, and the reported structures may have a significant impact on structure-based drug-design efforts. Comparisons with previously reported structures at lower resolution reveal the detailed conformation of important structural features in the allosteric transition of GP from the T-state to the R-state. The conformation of the N-terminal segment (residues 7-17), the position of which was not located in previous T-state structures, was revealed to form an α-helix (now termed α0). The conformation of this segment (which contains Ser14, phosphorylation of which leads to the activation of GP) is significantly different between the T-state and the R-state, pointing in opposite directions. In the T-state it is packed between helices α4 and α16 (residues 104-115 and 497-508, respectively), while in the R-state it is packed against helix α1 (residues 22'-38') and towards the loop connecting helices α4' and α5' of the neighbouring subunit. The allosteric binding site where AMP and IMP bind is formed by the ordering of a loop (residues 313-326) which is disordered in the free structure, and adopts a conformation dictated mainly by the type of nucleotide that binds at this site.
Subject(s)
Adenosine Monophosphate , Glycogen Phosphorylase , Muscles , Animals , Rabbits , Adenosine Monophosphate/metabolism , Allosteric Regulation , Crystallography, X-Ray , Glycogen Phosphorylase/chemistry , Glycogen Phosphorylase/metabolism , Models, Molecular , Muscle Proteins/chemistry , Muscle Proteins/metabolism , Muscles/enzymology , Protein Conformation , Substrate SpecificityABSTRACT
Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.
Subject(s)
Glucose/chemistry , Glucosephosphates/chemistry , Glycogen Phosphorylase/chemistry , Glycogen/chemistry , Amides/pharmacology , Animals , Caffeine/pharmacology , Ellagic Acid/pharmacology , Enzyme Assays , Glycogen Phosphorylase/antagonists & inhibitors , Glycogen Phosphorylase/isolation & purification , High-Throughput Screening Assays , Indoles/pharmacology , Kinetics , Rabbits , Solutions , Structure-Activity RelationshipABSTRACT
Glycogen debranching enzyme (GDE), together with glycogen phosphorylase (GP), is responsible for the complete degradation of glycogen. GDE has distinct catalytic sites for 4-α-glucanotransferase and amylo-α-1,6-glucosidase. For the GDE sensitive assay, we previously developed the GP limit fluorogenic branched dextrin Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4Glcα1-4GlcPA (B4/84, where Glc = D-glucose and GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol). However, B4/84 is not widely available because of difficulties in its chemical synthesis and positional-isomer separation (0.33% yield by α-1,6-coupling of maltotetraose with Glc7-GlcPA). In this study, we attempted to develop an efficient method for the preparation of Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-4Glcα1-4Glcα1-4Glcα1-6)Glcα1-4Glcα1-4GlcPA (B3/74), which was designed to have the minimum essential dextrin structure for GDE. First, Glcα1-6Glcα1-4Glcα1-4GlcPA (B3/31) was prepared from commercially available Glcα1-6Glcα1-4Glcα1-4Glc. Using α-cyclodextrin as a donor substrate, cyclodextrin glucanotransferase elongated both the main and side branches on B3/31, while all the glycosidic bonds in B3/31 were left intact. After exhaustive digestion with GP, B3/74 was obtained from B3/31 with 16% yield, a value that is 48-fold greater than that previously reported for B4/84. GDE 4-α-glucanotransferase exhibited high activity toward both B3/74 and B4/84. In addition, we studied the efficient conversion of B3/74 into Glcα1-4Glcα1-4Glcα1-4Glcα1-4(Glcα1-6)Glcα1-4Glcα1-4GlcPA (B3/71), which has the best dextrin structure for the GDE amylo-α-1,6-glucosidase.
Subject(s)
Dextrins/chemistry , Glycogen Debranching Enzyme System/chemistry , Glycogen/genetics , Liver/metabolism , Binding Sites/genetics , Chromatography, High Pressure Liquid , Fluorescent Dyes/chemistry , Glucosyltransferases/chemistry , Glycogen/chemistry , Glycogen Debranching Enzyme System/genetics , Glycogen Phosphorylase/chemistry , Glycogen Phosphorylase/genetics , Humans , Oligosaccharides/chemistryABSTRACT
Anthocyanins (ACNs) are dietary phytochemicals with an acknowledged therapeutic significance. Pomegranate juice (PJ) is a rich source of ACNs with potential applications in nutraceutical development. Glycogen phosphorylase (GP) catalyzes the first step of glycogenolysis and is a molecular target for the development of antihyperglycemics. The inhibitory potential of the ACN fraction of PJ is assessed through a combination of in vitro assays, ex vivo investigation in hepatic cells, and X-ray crystallography studies. The ACN extract potently inhibits muscle and liver isoforms of GP. Affinity crystallography reveals the structural basis of inhibition through the binding of pelargonidin-3-O-glucoside at the GP inhibitor site. The glucopyranose moiety is revealed as a major determinant of potency as it promotes a structural binding mode different from that observed for other flavonoids. This inhibitory effect of the ACN scaffold and its binding mode at the GP inhibitor binding site may have significant implications for future structure-based drug design endeavors.
Subject(s)
Anthocyanins/chemistry , Enzyme Inhibitors/chemistry , Fruit and Vegetable Juices/analysis , Glycogen Phosphorylase/chemistry , Plant Extracts/chemistry , Pomegranate/chemistry , Amino Acid Motifs , Animals , Binding Sites , Crystallography, X-Ray , Glycogen Phosphorylase/antagonists & inhibitors , Hep G2 Cells , Humans , Kinetics , Protein Binding , RabbitsABSTRACT
Glycogen constitutes the main store of glucose in animal cells. Being present at much lower concentrations in the brain than in liver and muscles, brain glycogen has long been considered as an emergency source of glucose, mobilized under stress conditions (including hypoglyceamia). Nevertheless, over the past decade, multiple studies have shed a new light on the roles of brain glycogen, being notably an energy supply critical for high-cognitive processes such as learning and memory consolidation. Glycogen phosphorylase (GP) is the key enzyme regulating the mobilization of glycogen in cells. It is found in humans as three isozymes: muscle (mGP), liver (lGP) and brain GP (bGP). In the brain, astrocytes express both mGP and bGP while neurons only express the brain isoform. Although GP isozymes are very similar, their distinct regulatory features confer them distinct metabolic functions that are strongly related to the roles of glycogen in different tissues. Here, we provide an overview of the functions, the regulations and the structures of GPs in the brain and their relation to the specific roles of glycogen in astrocytes and neurons. We also discuss novel findings concerning the specific regulations of bGP by oxidative stress, and the potential of these enzymes as therapeutic targets in the brain.
Subject(s)
Brain/enzymology , Glycogen Phosphorylase/chemistry , Glycogen Phosphorylase/metabolism , Glycogen , Animals , Brain/metabolism , Glycogen/metabolism , Humans , Isoenzymes/chemistry , Isoenzymes/metabolism , Liver/enzymology , Liver/metabolism , Muscles/enzymology , Muscles/metabolismABSTRACT
Epimeric series of aryl-substituted glucopyranosylidene-spiro-imidazolinones, an unprecedented new ring system, were synthesized from the corresponding Schiff bases of O-perbenzoylated (gluculopyranosylamine)onamides by intramolecular ring closure of the aldimine moieties with the carboxamide group elicited by N-bromosuccinimide in pyridine. Test compounds were obtained by Zemplén O-debenzoylation. Stereochemistry and ring tautomers of the new compounds were investigated by NMR, time-dependent density functional theory (TDDFT)-electronic circular dichroism, and DFT-NMR methods. Kinetic studies with rabbit muscle and human liver glycogen phosphorylases showed that the (R)-imidazolinones were 14-216 times more potent than the (S) epimers. The 2-naphthyl-substituted (R)-imidazolinone was the best inhibitor of the human enzyme (Ki 1.7 µM) and also acted on HepG2 cells (IC50 177 µM). X-ray crystallography revealed that only the (R) epimers bound in the crystal. Their inhibitory efficacy is based on the hydrogen-bonding interactions of the carbonyl oxygen and the NH of the imidazolinone ring.
Subject(s)
Enzyme Inhibitors/pharmacology , Glucosides/pharmacology , Glycogen Phosphorylase/antagonists & inhibitors , Imidazolines/pharmacology , Spiro Compounds/pharmacology , Animals , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , Glucosides/chemical synthesis , Glucosides/metabolism , Glycogen Phosphorylase/chemistry , Glycogen Phosphorylase/metabolism , Hep G2 Cells , Humans , Hydrogen Bonding , Imidazolines/chemical synthesis , Imidazolines/metabolism , Kinetics , Models, Molecular , Molecular Conformation , Protein Binding , Rabbits , Spiro Compounds/chemical synthesis , Spiro Compounds/metabolism , StereoisomerismABSTRACT
Glycogen phosphorylase (GP, EC 2.4.1.1) catalyze the rate-limiting step in glycogenolysis in animals, forming glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond. Therefore, GP plays a crucial role in carbohydrate metabolism. In the present study, the full-length cDNA sequence of GP (LvGP) was cloned from shrimp, Litopenaeus vannamei. The obtained 3242-bp LvGP cDNA sequence included a 5'-terminal untranslated region (UTR) of 48 bp, an open reading frame (ORF) of 2559 bp encoding a polypeptide of 852 amino acids (aa) and a 3'-UTR of 635 bp. The predicted LvGP protein sequence contained a typical phosphorylase domain (113-829 aa) and shared 72%-97% identities with GP from other species. Phylogenetic analysis revealed that LvGP showed the closest relationship with GP from Marsupenaeus japonicus. Tissue expression profiles showed that LvGP existed in most examined tissues, with the most predominant expression in the brain, followed by the muscles and stomach. LvGP transcripts in hepatopancreas and hemocytes were up regulated after the WSSV challenge. Furthermore, the role of LvGP in shrimp defending against WSSV infection was investigated by RNA interference (RNAi). Our findings showed that WSSV proliferation and shrimp accumulative mortality increased significantly after LvGP RNAi (Pâ¯<â¯0.01). The glycogen, glucose, and pyruvate content decreased in GP RNAi shrimp after WSSV injection, however, the lactate and ATP concentration enhanced. Moreover, lectin and anti-lipopolysaccharide factor2 (ALF2) were induced in LvGP silencing shrimp after WSSV infection, whereas the expression levels of crustin, ALF1 and ALF3 decreased. Our results suggested that the LvGP might play a crucial role in shrimp defending against WSSV infection by regulating metabolism and affecting the anti-infectious gene expression.
Subject(s)
Gene Expression Regulation/immunology , Glycogen Phosphorylase/genetics , Glycogen Phosphorylase/immunology , Immunity, Innate/genetics , Penaeidae/genetics , Penaeidae/immunology , Amino Acid Sequence , Animals , Arthropod Proteins/chemistry , Arthropod Proteins/genetics , Arthropod Proteins/immunology , Base Sequence , Gene Expression Profiling , Glycogen Phosphorylase/chemistry , Phylogeny , RNA Interference , Up-Regulation , White spot syndrome virus 1/physiologyABSTRACT
Glucopyranosylidene-spiro-benzo[ b][1,4]oxazinones were obtained via the corresponding 2-nitrophenyl glycosides obtained by two methods: (a) AgOTf-promoted glycosylation of 2-nitrophenol derivatives by O-perbenzoylated methyl (α-d-gluculopyranosyl bromide)heptonate or (b) Mitsunobu-type reactions of O-perbenzoylated methyl (α-d-gluculopyranose)heptonate with bulky 2-nitrophenols in the presence of diethyl azodicarboxylate (DEAD) and PPh3. Catalytic hydrogenation (H2-Pd/C) or partial reduction (e.g., H2-Pd/C, pyridine) of the 2-nitro groups led to spiro-benzo[ b][1,4]oxazinones and spiro-benzo[ b][1,4]-4-hydroxyoxazinones by spontaneous ring closure of the intermediate 2-aminophenyl or 2-hydroxylamino glycosides, respectively. The analogous 2-aminophenyl thioglycosides, prepared by reactions of O-perbenzoylated methyl (α-d-gluculopyranosyl bromide)heptonate with 2-aminothiophenols, were cyclized in m-xylene at reflux temperature to the corresponding spiro-benzo[ b][1,4]thiazinones. O-Debenzoylation was effected by Zemplén transesterification in both series. Spiro-configurations were determined by NMR and electronic circular dichroism time-dependent density functional theory (ECD-TDDFT) methods. Inhibition assays with rabbit muscle glycogen phosphorylase b showed (1' R)-spiro{1',5'-anhydro-d-glucitol-1',2-benzo[ b][1,4]oxazin-3(4 H)-one} and (1' R)-spiro{1',5'-anhydro-d-glucitol-1',2-benzo[ b][1,4]thiazin-3(4 H)-one} to be the most efficient inhibitors (27 and 28% inhibition at 625 µM, respectively). Plant growth tests with white mustard and garden cress indicated no effect except for (1' R)-4-hydroxyspiro{1',5'-anhydro-d-glucitol-1',2-benzo[ b][1,4]oxazin-3(4 H)-one} with the latter plant to show modest inhibition of germination (95% relative to control).
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Glycogen Phosphorylase/antagonists & inhibitors , Lepidium sativum/drug effects , Mustard Plant/drug effects , Spiro Compounds/chemical synthesis , Spiro Compounds/pharmacology , Animals , Enzyme Inhibitors/chemistry , Esterification , Germination/drug effects , Glycogen Phosphorylase/chemistry , Lepidium sativum/growth & development , Magnetic Resonance Spectroscopy , Molecular Structure , Mustard Plant/growth & development , Rabbits , Spiro Compounds/chemistry , Structure-Activity RelationshipABSTRACT
Structure-based design and synthesis of two biphenyl-N-acyl-ß-d-glucopyranosylamine derivatives as well as their assessment as inhibitors of human liver glycogen phosphorylase (hlGPa, a pharmaceutical target for type 2 diabetes) is presented. X-ray crystallography revealed the importance of structural water molecules and that the inhibitory efficacy correlates with the degree of disturbance caused by the inhibitor binding to a loop crucial for the catalytic mechanism. The in silico-derived models of the binding mode generated during the design process corresponded very well with the crystallographic data.
Subject(s)
Drug Design , Enzyme Inhibitors/chemistry , Glucosamine/analogs & derivatives , Glycogen Phosphorylase/chemistry , Quantitative Structure-Activity Relationship , Binding Sites , Catalytic Domain , Chemistry Techniques, Synthetic , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Glucosamine/chemical synthesis , Glucosamine/chemistry , Glucosamine/pharmacology , Glycogen Phosphorylase/antagonists & inhibitors , Humans , Hydrogen Bonding , Models, Molecular , Protein BindingABSTRACT
The interactome of arzanol was investigated by MS-based chemical proteomics, a pioneering technology for small molecule target discovery. Brain glycogen phosphorylase (bGP), a key regulator of glucose metabolism so far refractory to small molecule modulation, was identified as the main high-affinity target of arzanol. Competitive affinity-based proteomics, DARTS, molecular docking, surface plasmon resonance and in vitro biological assays provided molecular mechanistic insights into the arzanol-enzyme interaction, qualifying this positive modulator of bGP for further studies in the realm of neurodegeneration and cancer.
Subject(s)
Brain/enzymology , Glycogen Phosphorylase/metabolism , Phloroglucinol/analogs & derivatives , Pyrones/metabolism , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Binding Sites , Glycogen Phosphorylase/chemistry , HeLa Cells , Humans , Isoenzymes/chemistry , Isoenzymes/metabolism , Mass Spectrometry , Molecular Docking Simulation , Phloroglucinol/chemistry , Phloroglucinol/metabolism , Protein Structure, Tertiary , Proteomics , Pyrones/chemistry , Surface Plasmon ResonanceABSTRACT
A few new anthranilate diamide derivatives, 3aâ»e, 5aâ»c and 7aâ»d, were designed, synthesized, and evaluated for their inhibitory activity against two interesting antidiabetic targets, α-glucosidase and glycogen phosphorylase enzymes. Different instrumental analytical tools were applied in identification and conformation of their structures like; 13C NMR, ¹H NMR and elemental analysis. The screening of the novel compounds showed potent inhibitory activity with nanomolar concentration values. The most active compounds (5c) and (7b) showed the highest inhibitory activity against α-glucosidase and glycogen phosphorylase enzymes IC50 = 0.01247 ± 0.01 µM and IC50 = 0.01372 ± 0.03 µM, respectively. In addition, in vivo testing of the highly potent α-glucosidase inhibitor (7b) on rats with DTZ-induced diabetes was done and showed significant reduction of blood glucose levels compared to the reference drug. Furthermore, a molecular docking study was performed to help understand the binding interactions of the most active analogs with these two enzymes. The data obtained from the molecular modeling were correlated with those obtained from the biological screening. These data showed considerable antidiabetic activity for these newly synthesized compounds.
Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Glycogen Phosphorylase/antagonists & inhibitors , Glycoside Hydrolase Inhibitors/pharmacology , Hypoglycemic Agents/pharmacology , alpha-Glucosidases/chemistry , ortho-Aminobenzoates/pharmacology , Animals , Binding Sites , Blood Glucose/drug effects , Blood Glucose/metabolism , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/enzymology , Diabetes Mellitus, Experimental/physiopathology , Enzyme Assays , Glycogen Phosphorylase/chemistry , Glycogen Phosphorylase/metabolism , Glycoside Hydrolase Inhibitors/chemical synthesis , Hypoglycemic Agents/chemical synthesis , Male , Molecular Docking Simulation , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Rabbits , Rats , Rats, Sprague-Dawley , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/enzymology , Streptozocin , Structure-Activity Relationship , alpha-Glucosidases/metabolism , ortho-Aminobenzoates/chemical synthesisABSTRACT
The key regulatory enzymes of glycogenolysis are phosphorylase kinase, a hetero-oligomer with four different types of subunits, and glycogen phosphorylase, a homodimer. Both enzymes are activated by phosphorylation and small ligands, and both enzymes have distinct isoforms that are predominantly expressed in muscle, liver, or brain; however, whole-transcriptome high-throughput sequencing analyses show that in brain both of these enzymes are likely composed of subunit isoforms representing all three tissues. This Minireview examines the regulatory properties of the isoforms of these two enzymes expressed in the three tissues, focusing on their potential regulatory similarities and differences. Additionally, the activity, structure, and regulation of the remaining enzyme necessary for glycogenolysis, glycogen-debranching enzyme, are also reviewed.
Subject(s)
Brain/enzymology , Brain/metabolism , Glycogen Phosphorylase/metabolism , Glycogenolysis , Phosphorylase Kinase/metabolism , Animals , Energy Metabolism , Glycogen/metabolism , Glycogen Debranching Enzyme System/chemistry , Glycogen Debranching Enzyme System/metabolism , Glycogen Phosphorylase/chemistry , High-Throughput Screening Assays , Humans , Isoenzymes/metabolism , Ligands , Phosphorylase Kinase/chemistry , Phosphorylation , Structure-Activity Relationship , TranscriptomeABSTRACT
Pyridoxal 5'-phosphate (PLP)-dependent enzymes catalyze a wide range of reactions of amino acids and amines, with the exception of glycogen phosphorylase which exhibits peculiar both substrate preference and chemical mechanism. They represent about 4% of the gene products in eukaryotic cells. Although structure-function investigations regarding these enzymes are copious, their regulation by post-translational modifications is largely unknown. Protein phosphorylation is the most common post-translational modification fundamental in mediating diverse cellular functions. This review aims at summarizing the current knowledge on regulation of PLP enzymes by phosphorylation. Starting from the paradigmatic PLP-dependent glycogen phosphorylase, the first phosphoprotein discovered, we collect data in literature regarding functional phosphorylation events of eleven PLP enzymes belonging to different fold types and discuss the impact of the modification in affecting their activity and localization as well as the implications on the pathogenesis of diseases in which many of these enzymes are involved. The pivotal question is to correlate the structural consequences of phosphorylation among PLP enzymes of different folds with the functional modifications exerted in terms of activity or conformational changes or others. Although the literature shows that the phosphorylation of PLP enzymes plays important roles in mediating diverse cellular functions, our recapitulation of clue findings in the field makes clear that there is still much to be learnt. Besides mass spectrometry-based proteomic analyses, further biochemical and structural studies on purified native proteins are imperative to fully understand and predict how phosphorylation regulates PLP enzymes and to find the relationship between addition of a phosphate moiety and physiological response.
Subject(s)
Enzymes/chemistry , Enzymes/metabolism , Phosphates/metabolism , Pyridoxal Phosphate/metabolism , Amino Acids/metabolism , Glycogen Phosphorylase/chemistry , Glycogen Phosphorylase/metabolism , Humans , Models, Molecular , Phosphorylation , Protein Folding , Structure-Activity RelationshipABSTRACT
BACKGROUND: Glycogen phosphorylase (GP) is a pharmaceutical target for the discovery of new antihyperglycaemic agents. Punica granatum is a well-known plant for its potent antioxidant and antimicrobial activities but so far has not been examined for antihyperglycaemic activity. OBJECTIVE: The aim was to examine the inhibitory potency of eighteen polyphenolic extracts obtained from Punica granatum fruits and industrial juicing byproducts against GP and discover their most bioactive ingredients. METHOD: Kinetic experiments were conducted to measure the IC50 values of the extracts while affinity crystallography was used to identify the most bioactive ingredient. The inhibitory effect of one of the polyphenolic extracts was also verified ex vivo, in HepG2 cells. RESULTS: All extracts exhibited significant in vitro inhibitory potency (IC50 values in the range of low µg/mL). Affinity crystallography revealed that the most bioactive ingredients of the extracts were chlorogenic and ellagic acids, found bound in the active and the inhibitor site of GP, respectively.While ellagic acid is an established GP inhibitor, the inhibition of chlorogenic acid is reported for the first time. Kinetic analysis indicated that chlorogenic acid is an inhibitor with Ki=2.5 x 10-3Mthat acts synergistically with ellagic acid. CONCLUSION: Our study provides the first evidence for a potential antidiabetic usage of Punica granatum extracts as antidiabetic food supplements. Although, more in vivo studies have to be performed before these extracts reach the stage of antidiabetic food supplements, our study provides a first positive step towards this process.
Subject(s)
Fruit and Vegetable Juices , Glycogen Phosphorylase/antagonists & inhibitors , Hypoglycemic Agents/pharmacology , Lythraceae , Plant Extracts/pharmacology , Crystallography , Fruit , Glycogen Phosphorylase/chemistry , Glycogen Phosphorylase/metabolism , Hep G2 Cells , Humans , Plant Extracts/chemistryABSTRACT
The synthesized novel chloroquinoline derivatives 1-(2-chloro-4-phenylquinolin-3-yl)ethanone (CPQE), 1-(2,6-dichloro-4-phenylquinolin-3-yl)ethanone (DCPQE), methyl 2,6-dichloro-4-phenylquinoline-3-carboxylate (MDCPQC),methyl 2-chloro-4-methylquinoline-3-carboxylate (MCMQC) were subjected to the elementary analysis like FT-IR, NMR and Mass spectra using GCMS. Also, single crystal X-ray diffraction study was executed for the compound MDCPQC. The crystal packing is stabilized by C-H π and π-π interactions and also Chlorine-Chlorine short intermolecular contacts generating a three-dimensional supramolecular network. The antioxidant activity reduces high glucose level in the human body and hence the synthesized compounds were subjected for the estimation of antioxidant activity using DPPH method which exhibited good percentage of inhibition in comparison with ascorbic acid, a well-known anti-oxidant. The binding interaction of the chloroquinoline derivatives with calf thymus DNA (CT-DNA) has been explored by fluorescence quenching studies and molecular docking analysis has been employed to confirm the nature of binding. The prediction of pharmacological properties such as drug-likeness, molecular properties like absorption, distribution, metabolism, excretion and toxicity (ADMET) was carried out by computational studies to compare chloroquinoline derivatives with standard drug. Owing to the various potential biological activities of the quinoline compounds, molecular docking studies were also further carried out for the chloroquinoline derivatives, showing that they may act as effective anti-diabetic agents by inhibiting Glycogen Phosphorylase a protein.
Subject(s)
DNA/metabolism , Molecular Docking Simulation , Quinolines/chemical synthesis , Quinolines/pharmacology , Animals , Antioxidants/chemical synthesis , Antioxidants/chemistry , Antioxidants/metabolism , Antioxidants/pharmacology , Biphenyl Compounds/chemistry , Cattle , Chemistry Techniques, Synthetic , DNA/chemistry , Glycogen Phosphorylase/antagonists & inhibitors , Glycogen Phosphorylase/chemistry , Glycogen Phosphorylase/metabolism , Hypoglycemic Agents/chemical synthesis , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/metabolism , Hypoglycemic Agents/pharmacology , Nucleic Acid Conformation , Picrates/chemistry , Protein Conformation , Quinolines/chemistry , Quinolines/metabolism , RabbitsABSTRACT
Glycogen phosphorylase (GP) is an allosteric enzyme whose catalytic site comprises six subsites (SG1, SG-1, SG-2, SG-3, SG-4, and SP) that are complementary to tandem five glucose residues and one inorganic phosphate molecule, respectively. In the catalysis of GP, the nonreducing-end glucose (Glc) of the maltooligosaccharide substrate binds to SG1 and is then phosphorolyzed to yield glucose 1-phosphate. In this study, we probed the catalytic site of rabbit muscle GP using pyridylaminated-maltohexaose (Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA, where GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol]; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (Glc m -AltNAc-Glc n -GlcPA, where m + n = 4 and AltNAc is 3-acetoamido-3-deoxy-D-altrose). PA-0 served as an efficient substrate for GP, whereas the other PA-0 derivatives were not as good as the PA-0, indicating that substrate recognition by all the SG1 -SG-4 subsites was important for the catalysis of GP. By comparing the initial reaction rate toward the PA-0 derivatives (V derivative) with that toward PA-0 (V PA-0), we found that the value of V derivative/V PA-0 decreased significantly as the level of allosteric activation of GP increased. These results suggest that some conformational changes have taken place in the maltooligosaccharide-binding region of the GP catalytic site during allosteric regulation.
Subject(s)
Catalytic Domain , Glycogen Phosphorylase/chemistry , Glycogen Phosphorylase/metabolism , Oligosaccharides/metabolism , Adenosine Monophosphate/metabolism , Allosteric Regulation , Animals , Chromatography, High Pressure Liquid , Kinetics , Muscles/enzymology , Oligosaccharides/chemistry , Rabbits , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass SpectrometryABSTRACT
A 42-residue polypeptide conjugated to a small-molecule organic ligand capable of targeting the phosphorylated side chain of Ser15 was shown to bind glycogen phosphorylaseâ a (GPa) with a KD value of 280â nm. The replacement of hydrophobic amino acids by Ala reduced affinities, whereas the incorporation of l-2-aminooctanoic acid (Aoc) increased them. Replacing Nle5, Ile9 and Leu12 by Aoc reduced the KD value from 280 to 27â nm. "Downsizing" the 42-mer to an undecamer gave rise to an affinity for GPa an order of magnitude lower, but the undecamer in which Nle5, Ile9 and Leu12 were replaced by Aoc showed a KD value of 550â nm, comparable with that of the parent 42-mer. The use of Aoc residues offers a convenient route to increased affinity in protein recognition as well as a strategy for the "downsizing" of peptides essentially without loss of affinity. The results show that hydrophobic binding sites can be found on protein surfaces by comparing the affinities of polypeptide conjugates in which Aoc residues replace Nle, Ile, Leu or Phe with those of their unmodified counterparts. Polypeptide conjugates thus provide valuable opportunities for the optimization of peptides and small organic compounds in biotechnology and biomedicine.
Subject(s)
Glycogen Phosphorylase/chemistry , Hydrophobic and Hydrophilic Interactions , Peptides/chemistry , Binding Sites , Glycogen Phosphorylase/metabolism , Humans , Models, Molecular , Molecular Structure , Peptides/chemical synthesis , Peptides/metabolism , Protein Binding , Surface PropertiesABSTRACT
BACKGROUND: Mangiferin, was identified in the crude methanol extract, ethyl acetate, and n-butanol fractions of Aphloia theiformis (Vahl.) Benn. OBJECTIVE: This study aimed to analyze the plausible binding modes of mangiferin to key enzymes linked to diabetes type 2 (DT2), obesity, hypertension, Alzheimer's disease, and urolithiasis using molecular docking. METHOD: Crystallographic structures of α-amylase, α-glucosidase, glycogen phosphorylase (GP), pancreatic lipase, cholesterol esterase (CEase), angiotensin-I-converting enzyme (ACE), acetyl cholinesterase (AChE), and urease available on the Protein Databank database were docked to mangiferin using Gold 6.0 software. RESULTS: We showed that mangiferin bound to all enzymes by π-π and hydrogen bonds mostly. Mangiferin was docked to both allosteric and orthosteric sites of α-glucosidase by π-π interactions. However, several hydrogen bonds were observed at the orthosteric position, suggesting a preference for this site. The docking of mangiferin on AChE with the catalytic pocket occupied by paraoxon could be attributed to π-π stacking involving amino acid residues, Trp341 and Trp124. CONCLUSION: This study provided an insight of the molecular interaction of mangiferin with the studied enzymes and can be considered as a valuable tool for designing new drugs for better management of these diseases.