A novel inhibitor of glycogen phosphorylase from crayfish hepatopancreas

• 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
• 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
• 3.3. The inhibitor is a small protein (M_r = 23,000) which did not show proteolytic activity.
• 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
• 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
• 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.

     

The effects of isofagomine, a potent glycogen phosphorylase inhibitor, on glycogen metabolism in cultured mouse cortical astrocytes

A novel inhibitor of liver glycogen phosphorylase, isofagomine, was investigated as a possible inhibitor of the enzyme in the brain and in cultured astrocytes. Additionally, the effect of the drug on norepinephrine (NE) induced glycogen degradation in astrocytes was studied. Astrocytes were cultured from mouse cerebral cortex and homogenates were prepared from the cells as well as from mouse brain. Isofagomine dose-dependently inhibited glycogen phosphorylase when measured in the direction of glycogen degradation in both preparations with IC50 values (mean +/- SEM) of 1.0 +/- 0.1 microM and 3.3 +/- 0.5 microM in brain and astrocyte homogenates, respectively. Moreover, isofagomine at a concentration of 400 microM completely prevented NE induced depletion of glycogen stores and the concomitant lactate production in intact astrocytes. It is suggested that this novel glycogen phosphorylase inhibitor may be a valuable tool to investigate the functional importance of glycogen in astrocytes and in the brain.     

3,4-Dichloroisocoumarin, a serine protease inhibitor, inactivates glycogen phosphorylase b

3,4-Dichloroisocoumarin (3,4-DCI) is a highly reactive, mechanism-based inhibitor of serine proteases. We show here that glycogen phosphorylase b is also inactivated by this inhibitor, in a mechanism that parallels the inactivation of serine proteases, but involving multiple sites of covalent modification. Such a process may compromise studies in which 3,4-DCI is used to arrest proteolysis of a second native protein which may itself be modified.     

Synthesis of a C-glucosylated cyclopropylamide and evaluation as a glycogen phosphorylase inhibitor

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 2.5 mM).     

Evaluation of novel hyphodermin derivatives as glycogen phosphorylase a inhibitors

The lipophilicity, permeability, solubility, polar surface area and 'rule-of-five' properties were assessed, using QikProp v2.5 (Schr?dinger, Inc.) and ALOGPS 2.1 calculations, for 25 Hyphodermin derivatives. These compounds obeyed the 'rule-of-five', and the calculated physicochemical values were generally within desired limits. All compounds were tested against Glycogen Phosphorylase a (GPa). Four phenyl and benzyl substituted 2-oxo-hexahydro and tetrahydrobenzo[cd]indole carboxylic acids were identified as novel inhibitors of GPa with estimated IC(50) values in the range 0.8-1.3mM. Molecular modelling of these novel inhibitors was used to obtain the main structural features of this class of molecule for future structure-activity relationship studies.     

Integrated effects of multiple modulators on human liver glycogen phosphorylase a

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.     

Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site

Flavopiridol (L86-8275) ((-)-cis-5, 7-dihydroxy-2-(2-chlorophenyl)-8-[4-(3-hydroxy-1-methyl)-piperidinyl] -4H-benzopyran-4-one), a potential antitumor drug, currently in phase II trials, has been shown to be an inhibitor of muscle glycogen phosphorylase (GP) and to cause glycogen accumulation in A549 non-small cell lung carcinoma cells (Kaiser, A., Nishi, K., Gorin, F.A., Walsh, D.A., Bradbury, E. M., and Schnier, J. B., unpublished data). Kinetic experiments reported here show that flavopiridol inhibits GPb with an IC(50) = 15.5 microm. The inhibition is synergistic with glucose resulting in a reduction of IC(50) for flavopiridol to 2.3 microm and mimics the inhibition of caffeine. In order to elucidate the structural basis of inhibition, we determined the structures of GPb complexed with flavopiridol, GPb complexed with caffeine, and GPa complexed with both glucose and flavopiridol at 1.76-, 2.30-, and 2.23-A resolution, and refined to crystallographic R values of 0.216 (R(free) = 0.247), 0.189 (R(free) = 0.219), and 0.195 (R(free) = 0.252), respectively. The structures provide a rational for flavopiridol potency and synergism with glucose inhibitory action. Flavopiridol binds at the allosteric inhibitor site, situated at the entrance to the catalytic site, the site where caffeine binds. Flavopiridol intercalates between the two aromatic rings of Phe(285) and Tyr(613). Both flavopiridol and glucose promote the less active T-state through localization of the closed position of the 280s loop which blocks access to the catalytic site, thereby explaining their synergistic inhibition. The mode of interactions of flavopiridol with GP is different from that of des-chloro-flavopiridol with CDK2, illustrating how different functional parts of the inhibitor can be used to provide specific and potent binding to two different enzymes.     

Cardioprotective activity of a novel and potent competitive inhibitor of lactate dehydrogenase

Alkaline incubation of NADH results in the formation of a very potent inhibitor of lactate dehydrogenase. High resolution mass spectroscopy along with NMR characterization clearly showed that the inhibitor is derived from attachment of a glycolic acid moiety to the 4-position of the dihydronicotinamide ring of NADH. The very potent inhibitor is competitive with respect to NADH. The inhibitor added in submicromolar concentrations to cardiomyocytes protects them from damage caused by hypoxia/reoxygenation stress. In isolated mouse hearts, addition of the inhibitor results in a substantial reduction of myocardial infarct size caused by global ischemia/reperfusion injury.     

Crystallographic studies on N-azidoacetyl-beta-D-glucopyranosylamine, an inhibitor of glycogen phosphorylase: comparison with N-acetyl-beta-D-glucopyranosylamine

N-acetyl-beta-D-glucopyranosylamine (NAG) is a potent inhibitor (Ki=32 microM) of glycogen phosphorylase b (GPb), and has been employed as a lead compound for the structure-based design of new analogues, in an effort to utilize its potential as a hypoglycaemic agent. Replacement of the acetamido group by azidoacetamido group resulted in an inhibitor, N-azidoacetyl-beta-D-glucopyranosylamine (azido-NAG), with a Ki value of 48.7 microM, in the direction of glycogen synthesis. In order to elucidate the mechanism of inhibition, we determined the ligand structure in complex with GPb at 2.03 A resolution, and the structure of the fully acetylated derivative in the free form. The molecular packing of the latter is stabilized by a number of bifurcated hydrogen bonds of which the one involving a bifurcated C-H...N...H-C type hydrogen bonding is rather unique in organic azides. Azido-NAG can be accommodated in the catalytic site of T-state GPb at approximately the same position as that of NAG and stabilizes the T-state conformation of the 280 s loop by making several favourable contacts to residues of this loop. The difference observed in the Ki values of the two analogues can be interpreted in terms of desolvation effects, subtle structural changes of protein residues and changes in water structure.     

The cyclin-dependent kinase (CDK) inhibitor flavopiridol inhibits glycogen phosphorylase

Flavopiridol has been shown to induce cell cycle arrest and apoptosis in various tumor cells in vitro and in vivo. Using immobilized flavopiridol, we identified glycogen phosphorylases (GP) from liver and brain as flavopiridol binding proteins from HeLa cell extract. Purified rabbit muscle GP also bound to the flavopiridol affinity column. GP is the rate-limiting enzyme in intracellular glycogen breakdown. Flavopiridol significantly inhibited the AMP-activated GP-b form of the purified rabbit muscle isoenzyme (IC50 of 1 microM at 0.8 mM AMP), but was less inhibitory to the active phosphorylated form of GP, GP-a (IC50 of 2.5 microM). The AMP-bound GP-a form was poorly inhibited by flavopiridol (40% at 10 microM). Increasing concentrations of the allosteric effector AMP resulted in a linear decrease in the GP-inhibitory activity of flavopiridol suggesting interference between flavopiridol and AMP. In contrast the GP inhibitor caffeine had no effect on the relative GP inhibition by flavopiridol, suggesting an additive effect of caffeine. Flavopiridol also inhibited the phosphorylase kinase-catalyzed phosphorylation of GP-b by inhibiting the kinase in vitro. Flavopiridol thus is able to interfere with both activating modifications of GP-b, AMP activation and phosphorylation. In A549 NSCLC cells flavopiridol treatment caused glycogen accumulation despite of an increase in GP activity, suggesting direct GP inhibition in vivo rather than inhibition of GP activation by phosphorylase kinase. These results suggest that the cyclin-dependent kinase inhibitor flavopiridol interferes with glycogen degradation, which may be responsible for flavopiridol's cytotoxicity and explain its resistance in some cell lines.     

Intensity and duration effects of exercise on heart cAMP, phosphorylase, and glycogen

Glycogen, phosphorylase, and adenosine 3',5'-cyclic monophosphate (cAMP) were determined in rat heart following an acute exercise bout. Intensity and duration of exercise were varied to gain further insights into the mechanism regulating myocardial glycogenolysis during exercise. Groups of rats were run at either 15 or 30 m/min for 0, 5, 10, 15, or 30 min and immediately killed. Heart glycogen degradation was influenced by intensity and duration of exercise and was independent of cAMP levels and activation of phosphorylase to its a form. cAMP levels were increased in the heart, dependent on intensity and duration of exercise. Phosphorylase in the a form increased at the onset of exercise, independent of intensity, and remained elevated throughout the exercise despite little or no glycogenolysis. Absolute phosphorylase a activity was also increased with exercise and was independent of intensity of exercise. Compared with resting levels, total phosphorylase activity was decreased at all times at the lower exercise intensity, whereas total phosphorylase activity declined at the higher intensity only after glycogenolysis had occurred. These data suggest that myocardial glycogen degradation during exercise can occur independently of cAMP and that the percentage of phosphorylase in the a form is not a good indicator of glycogenolytic rate.     

Glycogenolytic, noninsulin-like effects of vanadate on rat hepatocyte glycogen synthase and phosphorylase

Vanadate inactivated rat hepatocyte glycogen synthase and activated glycogen phosphorylase in a dose- and time-dependent manner. These effects were observed in hepatocytes from both fasted as well as fed rats. When rat hepatocytes were preincubated with [32P]phosphate and then with vanadate, and the 32P-labeled glycogen synthase was specifically immunoprecipitated, it was observed that vanadate stimulated the phosphorylation of the 88,000-dalton subunit of glycogen synthase. All of the phosphate was located in the same two CNBr fragments of the enzyme which are phosphorylated by glucagon and other glycogenolytic hormones. In cells incubated in a calcium-depleted medium, vanadate was still able to inactivate glycogen synthase but its effects on phosphorylase were essentially lost. These results demonstrate that, in the hepatocyte, vanadate exerts opposite effects than in the adipocyte and skeletal muscle, where vanadate has an insulin-like action.     

A tentative mechanism of the ternary complex formation between phosphorylase kinase, glycogen phosphorylase b and glycogen

The kinetics of rabbit skeletal muscle phosphorylase kinase interaction with glycogen has been studied. At pH 6.8 the binding of phosphorylase kinase to glycogen proceeds only in the presence of Mg2+, whereas at pH 8.2 formation of the complex occurs even in the absence of Mg2+. On the other hand, the interaction of phosphorylase kinase with glycogen requires Ca2+ at both pH values. The initial rate of the complex formation is proportional to the enzyme and glycogen concentrations, suggesting the formation of the complex with stoichiometry 1:1 at the initial step of phosphorylase kinase binding by glycogen. According to the kinetic and sedimentation data, the substrate of the phosphorylase kinase reaction, glycogen phosphorylase b, favors the binding of phosphorylase kinase with glycogen. We suggest a model for the ordered binding of phosphorylase b and phosphorylase kinase to the glycogen particle that explains the increase in the tightness of phosphorylase kinase binding with glycogen in the presence of phosphorylase b.     

Schistosoma haematobium: histochemistry of glycogen, glycogen phosphorylase a and glycogen branching enzyme in niridazole-treated females.

The body posterior to the ovary of Schistosoma haematobium females was investigated. Glycogen, glycogen phosphorylase a (EC 2.4.1.1) and glycogen branching enzyme (EC 2.4.1.18) activities were detected in the subtegumental muscle system, parenchyma and mature vitelline cells, whereas no activities were detected in the tegument and immature vitelline cells of the parasite. Administration of a single niridazole dose of 250 mg kg-1 to the pouched mouse (Saccostomus camestris) produced the following changes in S. haematobium females: a relatively rapid depletion of glycogen stores due to disruption of the absorptive surface of the parasite, and to an increase in the activity of glycogen phosphorylase a; a reduction in the phosphorylase a to phosphorylase b-conversion capacity of glycogen phosphorylase phosphatase (EC 3.1.3.17); a decrease in glycogen branching enzyme activity; and a relatively rapid degeneration of parasite cells possibly due to their loss of endogenous energy reserves.     

Anti-angiogenic activity of a novel multi-substrate analogue inhibitor of thymidine phosphorylase.

7-Deazaxanthine (7-DX) was recently identified as the first purine derivative with pronounced inhibitory activity against Escherichia coli thymidine phosphorylase (TP) and angiogenesis. In order to "freeze" the enzyme in an open, inactive conformation, a novel multi-substrate analogue inhibitor of TP, containing an alkyl phosphonate moiety covalently linked to 7-DX, was synthesized. The prototype compound TP65 (9-(8-phosphonooctyl)-7-deazaxanthine) (at 250 microM) completely inhibited TP-induced formation of microvascular sprouts from endothelial cell aggregates in a three-dimensional fibrin gel. In the chick chorioallantoic membrane assay, TP caused a dose-dependent stimulation of angiogenesis, which was completely inhibited by 250 nmol TP65. This dose proved to be non-toxic for the developing chick embryo. TP65 thus emerges as a potent and specific inhibitor of TP and TP-induced angiogenesis, which opens new perspectives for multi-substrate analogue inhibitors of TP as potential anti-cancer agents and as inhibitors of angiogenesis and of diseases with enhanced expression of TP.     

Interaction between glycogen and glycogen phosphorylase of Tetrahymena

The glycogen phosphorylase of Tetrahymena pyriformis complexes with glycogen as judged by its elution pattern from columns of Sepharose 6B. Complex formation does not occur with starch, amylose, or amylopectin, and neither do these polyglucans serve as primers for the enzyme. To study the association between the phosphorylase and glycogen particles in situ, Tetrahymena were grown under differing physiological conditions, phosphorylase was isolated and chromatographed on a Sepharose 6B column. Phosphorylase activity isolated from cells grown in the absence of glucose was only partially associated with glycogen, while in cells exposed to glucose for 30 min or more all the phosphorylase activity was associated with glycogen. The effects of culture age and anaerobiosis on the relative amounts of free and glycogen-bound enzyme in the cells were also studied. It was concluded from the in vivo experiments that there was no simple relation between the fraction of enzyme bound to glycogen and between cell glycogen content.     

Hexokinase 2, glycogen synthase and phosphorylase play a key role in muscle glycogen supercompensation

Background

Glycogen-depleting exercise can lead to supercompensation of muscle glycogen stores, but the biochemical mechanisms of this phenomenon are still not completely understood.

Methods

Using chronic low-frequency stimulation (CLFS) as an exercise model, the tibialis anterior muscle of rabbits was stimulated for either 1 or 24 hours, inducing a reduction in glycogen of 90% and 50% respectively. Glycogen recovery was subsequently monitored during 24 hours of rest.

Results

In muscles stimulated for 1 hour, glycogen recovered basal levels during the rest period. However, in those stimulated for 24 hours, glycogen was supercompensated and its levels remained 50% higher than basal levels after 6 hours of rest, although the newly synthesized glycogen had fewer branches. This increase in glycogen correlated with an increase in hexokinase-2 expression and activity, a reduction in the glycogen phosphorylase activity ratio and an increase in the glycogen synthase activity ratio, due to dephosphorylation of site 3a, even in the presence of elevated glycogen stores. During supercompensation there was also an increase in 5′-AMP-activated protein kinase phosphorylation, correlating with a stable reduction in ATP and total purine nucleotide levels.

Conclusions

Glycogen supercompensation requires a coordinated chain of events at two levels in the context of decreased cell energy balance: First, an increase in the glucose phosphorylation capacity of the muscle and secondly, control of the enzymes directly involved in the synthesis and degradation of the glycogen molecule. However, supercompensated glycogen has fewer branches.