Anthranilimide-based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes: 1. Identification of 1-amino-1-cycloalkyl carboxylic acid headgroups

Optimization of the amino acid residue within a series of anthranilimide-based glycogen phosphorylase inhibitors is described. These studies culminated in the identification of anthranilimides 16 and 22 which displayed potent in vitro inhibition of GPa in addition to reduced inhibition of CYP2C9 and excellent pharmacokinetic properties.     

Synthetic studies in butenonyl C-glycosides: Preparation of polyfunctional alkanonyl glycosides and their enzyme inhibitory activity

A simple synthesis of phenyl butenoyl C-glycosides has been achieved by Aldol condensation of peracetylated glycosyl acetones with aromatic aldehydes followed by deacetylation with methanolic NaOMe. The selected butenoyl C-glycosides on conjugate addition of diethyl malonate resulted in polyfunctional alkanonyl glycosides in good yields. The butenoyl C- and alkanoyl C-glycosides were evaluated for their α-glucosidase, glucose-6-phosphatse and glycogen phosphorylase enzyme inhibitory activities in vitro. Three of the synthesized (3, 5 and 9) showed potent enzyme inhibitory activities as compared to standard drugs. Compounds 3, 5 and 9 were evaluated in vivo too displaying significant activity as compared to standard drugs acarbose and metformin.     

Role of glucose and insulin in regulating glycogen synthase and phosphorylase activities in rainbow trout hepatocytes

This study, using ¹³C nuclear magnetic resonance spectroscopy showed enrichment of glycogen carbon (C1) from ¹³C-labelled (C1) glucose indicating a direct pathway for glycogen synthesis from glucose in rainbow trout (Oncorhynchus mykiss) hepatocytes. There was a direct relationship between hepatocyte glycogen content and total glycogen synthase, total glycogen phosphorylase and glycogen phosphorylase a activities, whereas the relationship was inverse between glycogen content and % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio. Incubation of hepatocytes with glucose (3 or 10 mmol·1^-1) did not modify either glycogen synthase or glycogen phosphorylase activities. Insulin (porcine, 10^-8 mol·1^-1) in the medium significantly decreased total glycogen phosphorylase and glycogen phosphorylase a activities, but had no significant effect on glycogen synthase activities when compared to the controls (absence of insulin). In the presence of 10 mmol·1^-1 glucose, insulin increased % glycogen synthase a and decreased % glycogen phosphorylase a activities in trout hepatocytes. Also, the effect of insulin on the activities of % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio were more pronounced at low than at high hepatocyte glycogen content. The results indicate that in trout hepatocytes both the glycogen synthetic and breakdown pathways are active concurrently in vitro and any subtle alterations in the phosphorylase to synthase ratio may determine the hepatic glycogen content. Insulin plays an important role in the regulation of glycogen metabolism in rainbow trout hepatocytes. The effect of insulin on hepatocyte glycogen content may be under the control of several factors, including plasma glucose concentration and hepatocyte glycogen content.     

5,6-Diarylanthranilo-1,3-dinitriles as a new class of antihyperglycemic agents

Various functionalized mono- and diarylanthranilo-1,3-dinitriles were synthesized and evaluated for their in vitro antihyperglycemic activity against the PTP-1B, glucose-6-phosphatase, glycogen phosphorylase and α-glucosidase enzymes. Among various screened compounds, 5,6-diaryl substituted anthranilo-1,3-dinitriles 3a, 3b, and 3d showed good inhibitory activity against PTP-1B with IC₅₀ values of 58–72 μM. Three of the test compounds showed significant (25–37%) lowering of plasma glucose level at 24 h in sucrose-challenged streptozotocin-induced diabetic Sprague–Dawley rat model.     

Anthranilimide-based glycogen phosphorylase inhibitors for the treatment of Type 2 diabetes: 2. Optimization of serine and threonine ether amino acid residues

Optimization of the amino acid residue of a series of anthranilimide-based glycogen phosphorylase inhibitors is described leading to the identification of serine and threonine ether analogs. t-Butylthreonine analog 20 displayed potent in vitro inhibition of GPa, low potential for P450 inhibition, and excellent pharmacokinetic properties.     

Synthesis,in vitro ADME profiling and in vivo pharmacological evaluation of novel glycogen phosphorylase inhibitors

A small set of indole-2-carboxamide derivatives identified from a high-throughput screening campaign has been described as a novel, potent, and glucose-sensitive inhibitors of glycogen phosphorylase a (GPa). Among this series of compounds, compound 2 exhibited moderate GP inhibitory activity (IC₅₀ = 0.29 μM), good cellular efficacy (IC₅₀ = 3.24 μM for HepG2 cells and IC₅₀ = 7.15 μM for isolated rat hepatocytes), together with good absorption, distribution, metabolism, and elimination (ADME) profiles. The in vivo animal study revealed that compound 2 significantly inhibited an increase of fasting blood glucose level in adrenaline-induced diabetic mice.     

Calcium-dependent regulation of glycogen synthase activity in a muscle glycogen particle

The calcium-dependent inactivation of glycogen synthase in an isolated glycogen-protein complex (glycogen pellet) from rabbit skeletal muscle has been investigated. Addition of 1 mm Ca²⁺, 10 mm Mg²⁺, and 1 mm ATP-γ-S to a concentrated suspension of glycogen pellet resulted in a rapid activation of glycogen phosphorylase concomitant with an inactivation of glycogen synthase. These conversion reactions were blocked by ethylene glycol bis(β-aminoethyl ether) N, N′-tetraacetic acid or by pretreatment of the complex with an antiserum to purified phosphorylase kinase. These data suggest that in the glycogen-protein complex, which may be a functional unit of glycogen metabolism in vivo, phosphorylase kinase can catalyze a Ca²⁺-dependent activation of glycogen phosphorylase synchronized with an inactivation of glycogen synthase. If under similar conditions phosphoprotein phosphatase activity was assayed using exogenous [³²P]phosphorylase, there was an apparent inactivation of the phosphatase. Evidence is presented that this apparent inactivation of phosphatase was due to an accumulation of endogenous phosphorylase a which acted as an inhibitor to the exogenous [³²P]-phosphorylase.     

Regulation of glycogen metabolism in the adrenal gland. IV. The effect of insulin on glycogen synthetase, phosphorylase, and related metabolites

Previous studies have indicated that the glycogen content of adrenal glands of fasted rats can be depleted by insulin per se (Bindstein, E., Piras, R., and Piras, M. M., Endocrinology88, 223, 1971). In order to establish the mechanism of action of this hormone in the adrenal gland, the effect of insulin has been now investigated on glycogen synthetase (UDP-glucose: α-1,4 glucan α-4-glueosyl-transferase, EC 2.4.1.11), glycogen phosphorylase (α-1,4 glucan: orthophosphate glucosyl-transferase, EC 2.4.1.1) and metabolites related to these enzymes.Approximately 40% of total adrenal glycogen phosphorylase of fasted rats is in the active form, which increases to 75% 1 hr after insulin treatment (75 mU/100 g body wt). This conversion occurs without apparent large changes of 3′-5′ cyclic AMP. Concomitantly with the enzymatic change, the levels of glucose-6-P, UDP-glucose and P_i suffer alterations which favor an increased phosphorolytic activity during the first hour of insulin treatment. Glycogen synthetase, which did not change during this period, is converted to the glucose-6-P independent form during the 2–3 hr of treatment. This conversion is preceded by an increased glycogen synthetase phosphatase activity, which seems to follow an inverse relationship with the glycogen level.The results obtained suggest that the effect of insulin on the adrenal gland of fasted rats is glycogenolytic, that is, opposite to that described for this hormone in other normal tissues. The glycogen depletion, on the other hand, seems to set in motion the mechanism for glycogen synthetase activation, with the subsequent glycogen resynthesis.     

Identification of 1,2,4-triazoles as new thymidine phosphorylase inhibitors: Future anti-tumor drugs

Thymidine phosphorylase (TP) is over expressed in several solid tumors and its inhibition can offer unique target suitable for drug discovery in cancer. A series of 1,2,4-triazoles 3a–3l has been synthesized in good yields and subsequently inhibitory potential of synthesized triazoles 3a–3l against thymidine phosphorylase enzyme was evaluated. Out of these twelve analogs five analogues 3b, 3c, 3f, 3l and 3l exhibited a good inhibitory potential against thymidine phosphorylase. Inhibitory potential in term of IC₅₀ values were found in the range of 61.98 ± 0.43 to 273.43 ± 0.96 μM and 7-Deazaxanthine was taken as a standard inhibitor with IC₅₀ = 38.68 ± 4.42 μM. Encouraged by these results, more analogues 1,2,4-triazole-3-mercaptocarboxylic acids 4a–4g were synthesized and their inhibitory potential against thymidine phosphorylase was evaluated. In this series, six analogues 4b–4g exhibited a good inhibitory potential in the range of 43.86 ± 1.11–163.43 ± 2.03 μM. Angiogenic response of 1,2,4-triazole acid 4d was estimated using the chick chorionic allantoic membrane (CAM) assay. In the light of these findings, structure activity relationship and molecular docking studies of selected triazoles to determine the key binding interactions was discussed. Docking studies demonstrate that synthesized analogues interacted with active site residues of thymidine phosphorylase enzyme through π-π stacking, thiolate and hydrogen bonding interactions.     

Anthranilimide based glycogen phosphorylase inhibitors for the treatment of type 2 diabetes. Part 3: X-ray crystallographic characterization,core and urea optimization and in vivo efficacy

Key binding interactions of the anthranilimide based glycogen phosphorylase a (GPa) inhibitor 2 from X-ray crystallography studies are described. This series of compounds bind to the AMP site of GP. Using the binding information the core and the phenyl urea moieties were optimized. This work culminated in the identification of compounds with single nanomolar potency as well as in vivo efficacy in a diabetic model.     

Purification of glycogen phosphorylase b from AMP-aminohydrolase activity

The presence of AMP aminohydrolase (EC 3.5.4.6) activity in glycogen phosphorylase b (EC 2.4.1.1) preparations, suggested by L. N. Johnson, N. B. Madsen, J. Mosley, and K. S. Wilson (1974, J. Mol. Biol.90, 703–717), has been confirmed in our laboratory. Since the hydrolase catalyzes the conversion of AMP into IMP the presence of traces of this impurity would dramatically affect, and could even invalidate, the results concerning some studies on the phosphorylase b-AMP interaction. The incubation of the phosphorylase b preparations with alumina Cγ in the cold for a brief period of time is proposed as a simple method of efficiently eliminating this impurity.     

Design and synthesis of 2,4-disubstituted polyhydroquinolines as prospective antihyperglycemic and lipid modulating agents

A series of 2,4-disubstituted polyhydroquinoline were synthesized and evaluated for their in vivo antihyperglycemic as well as antidyslipidemic activities. Several synthesized compounds have exhibited promising in vivo antihyperglycemic in SLM, STZ-S, and db/db mice model along with significant lipid and TG modulating activity. All these compounds were evaluated in various in vitro models of diabetes to know the possible mechanism of their antihyperglycemic action. Interestingly, compounds 3a–r (diaryl substitution) have exhibited promising protein-tyrosine phosphatase 1B (PTP1B) inhibitory activity whereas, compounds 5a–d (acid substituted) have shown significant glycogen phosphorylase activity.     

Wine Yeast Strains Engineered for Glycogen Overproduction Display Enhanced Viability under Glucose Deprivation Conditions

We used metabolic engineering to produce wine yeasts with enhanced resistance to glucose deprivation conditions. Glycogen metabolism was genetically modified to overproduce glycogen by increasing the glycogen synthase activity and eliminating glycogen phosphorylase activity. All of the modified strains had a higher glycogen content at the stationary phase, but accumulation was still regulated during growth. Strains lacking GPH1, which encodes glycogen phosphorylase, are unable to mobilize glycogen. Enhanced viability under glucose deprivation conditions occurs when glycogen accumulates in the strain that overexpresses GSY2, which encodes glycogen synthase and maintains normal glycogen phosphorylase activity. This enhanced viability is observed under laboratory growth conditions and under vinification conditions in synthetic and natural musts. Wines obtained from this modified strain and from the parental wild-type strain don't differ significantly in the analyzed enological parameters. The engineered strain might better resist some stages of nutrient depletion during industrial use.     

Zum histochemischen Phosphorylasenachweis im Herzinfarkt

Summary The demonstration of glycogen by PAS-method and the reaction of phosphorylase by Takeuchi et al. were performed in the hearts of rats 30 min after experimental coronary occlusion. While the cardiac muscle cells in the center of the ischemic region still contained glycogen, the cells in the border zone of the infarction did not. The phosphorylase activity was demonstrable only in those cardiac muscle cells which contained glycogen. As well known that the demonstration of phosphorylase activity is possible only if glycogen as starter is present, it is concluded that the lack of phosphorylase activity by the histochemical reaction in cells which do not contain glycogen doesn't prove the absence of the enzyme.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Stipendiat der Alexander von Humboldt-Stiftung.     

Dephosphorylation of skeletal muscle phosphorylase,glycogen synthase,and phosphorylase kinase β-subunit by a Mn2+-activated protein phosphatase

An Mn²⁺-activated phosphoprotein phosphatase of M_r = 80,000 from rabbit muscle catalyzes the dephosphorylation of skeletal muscle proteins that are phosphorylated by either phosphorylase kinase or cAMP-dependent protein kinase. Phosphorylase or glycogen synthase labeled by phosphorylase kinase at seryl residues 14 or 7, respectively, are both dephosphorylated by the phosphatase. Phosphorylase a and glycogen synthase compete with one another for the phosphatase. The phosphatase discriminates between different sites labeled by the cAMP-dependent protein kinase: glycogen synthase phosphorylated either to 1.0 or 1.8 mol phosphate/mol, or phosphorylase kinase phosphorylated on its β-subunit serve as substrates for the phosphatase, but the phosphorylase kinase α-subunit, the phosphorylated phosphatase inhibitor 1, or casein do not. Histone fraction IIA, phosphorylated by the catalytic subunit, was a poor substrate even at a concentration of 100 μm. Phosphorylation of the α-subunit of phosphorylase kinase had no influence on the kinetics of dephosphorylation of the β-subunit. Thus, the M_r = 80,000 phosphatase meets the functional definition of a protein phosphatase 1 [Cohen, P. (1978) Curr. Top. Cell. Regul.14, 117–196]. Furthermore, from a comparison of the known phosphorylated sites of these proteins, it appears that the phosphatase discriminates between different sites present in the phosphoproteins tested on the basis of the K_m values for the reactions. It displays a preferential activity toward proteins with a primary structure wherein basic residues are two positions amino-terminal from the phosphoserine, AgrLysX-YSer(P) or LysArgX-YSer(P), rather and one residue away, ArgArgX-Ser(P).     

Terpenoids. III: Synthesis and biological evaluation of 23-hydroxybetulinic acid derivatives as novel inhibitors of glycogen phosphorylase

A series of 23-hydroxybetulinic acid derivatives were prepared and tested in vitro as a new class of inhibitors of glycogen phosphorylase (GP). Within this series of compounds, 12b (IC₅₀ = 3.5 μM) is the most potent GPa inhibitor. The preliminary SAR results of the 23-hydroxybetulinic acid derivatives are discussed.     

1-(3-Deoxy-3-fluoro-β-d-glucopyranosyl) pyrimidine derivatives as inhibitors of glycogen phosphorylase b: Kinetic,crystallographic and modelling studies

Design of inhibitors of glycogen phosphorylase (GP) with pharmaceutical applications in improving glycaemic control in type 2 diabetes is a promising therapeutic strategy. The catalytic site of muscle glycogen phosphorylase b (GPb) has been probed with five deoxy-fluro-glucose derivatives. These inhibitors had fluorine instead of hydroxyl at the 3′ position of the glucose moiety and a variety of pyrimidine derivatives at the 1′ position. The best of this carbohydrate-based family of five inhibitors displays a K_i value of 46 μM. To elucidate the mechanism of inhibition for these compounds, the crystal structures of GPb in complex with each ligand were determined and refined to high resolution. The structures demonstrated that the inhibitors bind preferentially at the catalytic site and promote the less active T state conformation of the enzyme by making several favorable contacts with residues of the 280s loop. Fluorine is engaged in hydrogen bond interactions but does not improve glucose potency. The pyrimidine groups are located between residues 284–286 of the 280s loop, Ala383 of the 380s loop, and His341 of the β-pocket. These interactions appear important in stabilizing the inactive quaternary T state of the enzyme. As a follow up to recent computations performed on β-d-glucose pyrimidine derivatives, tautomeric forms of ligands 1–5 were considered as potential binding states. Using Glide-XP docking and QM/MM calculations, the ligands 2 and 5 are predicted to bind in different tautomeric states in their respective GPb complexes. Also, using α-d-glucose as a benchmark model, a series of substitutions for glucose –OH at the 3′ (equatorial) position were investigated for their potential to improve the binding affinity of glucose-based GPb catalytic site inhibitors. Glide-XP and quantum mechanics polarized ligand (QPLD-SP/XP) docking calculations revealed favorable binding at this position to be dominated by hydrogen bond contributions; none of the substitutions (including fluorine) out-performed the native –OH substituent which can act both as hydrogen bond donor and acceptor. The structural analyses of these compounds can be exploited towards the development of better inhibitors.     

Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused 2-aminopyrimidines as antitubercular and antidiabetic agents

An economical and efficient one step synthesis of a series of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydro-quinazolin-2-ylamines and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylamines by the reaction of bis-benzylidene cycloalkanones and guanidine hydrochloride in presence of NaH has been developed. All the synthesized compounds were evaluated against Mycobacterium tuberculosis H₃₇Rv strain and the α-glucosidase and glycogen phosphorylase enzymes. Few of the compounds have shown interesting in vitro activity with MIC up to 3.12 μg/mL against M. tuberculosis and very good inhibition of α-glucosidase and glycogen phosphorylase enzymes. The most potent non toxic compound 40 exhibited about 58% ex vivo activity at MIC of 3.12 μg/mL. The present study opens a new gate to synthesize antitubercular agents for diabetic TB patients. In silico docking studies indicate that mycobacterial dihydrofolate reductase is the possible target of these compounds.     

Regulation of fat body glycogen phosphorylase in larval tobacco hornworm,Manduca sexta

Activation and inactivation of fat body glycogen phosphorylase was investigated in ligated abdomens of larval Manduca sexta and in vitro. After maximal activation through Manduca adipokinetic hormone (AKH) or chilling, inactivation of glycogen phosphorylase commenced as soon as the stimulus for the activation was removed indicating that the enzyme system in the fat body is fine-tuned to low phosphorylase activities which is necessary to allow glycogen synthesis. In intact ligated abdomens phosphorylase can be activated repeatedly by either stimulus showing that the fat body system does not lose its responsiveness. It was impossible to achieve complete conversion of the inactive form of phosphorylase into the active form even after administration of AKH and simultaneous chilling. © 1992 Wiley-Liss, Inc.     

Adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscle with increased glycogen content

The effect of glycogen content on the activation of glycogen phosphorylase during adrenaline stimulation was investigated in soleus muscles from Wistar rats. Furthermore, adrenergic activation of glycogen phosphorylase in the slow-twitch oxidative soleus muscle was compared to the fast-twitch glycolytic epitrochlearis muscle. The glycogen content was 96.4 +/- 4.4 mmol (kg dw)(-1) in soleus muscles. Three hours of incubation with 10 mU/ml of insulin (and 5.5 mM glucose) increased the glycogen content to 182.2+/-5.9 mmol (kg dw)(-1) which is similar to that of epitrochlearis muscles (175.7+/-6.9 mmol (kg dw)(-1)). Total phosphorylase activity in soleus was independent of glycogen content. Adrenaline (10(-6) M) transformed about 20% and 35% (P < 0.01) of glycogen phosphorylase to the a form in soleus with normal and high glycogen content, respectively. In epitrochlearis, adrenaline stimulation transformed about 80% of glycogen phosphorylase to the a form. Glycogen synthase activation was reduced to low level in soleus muscles with both normal and high glycogen content. In conclusion, adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscles with increased glycogen content. Glycogen phosphorylase activation during adrenaline stimulation was much higher in epitrochlearis than in soleus muscles with a similar content of glycogen.