Search

Glycogen phosphorylase (GP) is the rate-determining enzyme in glycogenolysis, and its druggability has been extensively studied over the years for the development of therapeutics against type 2 diabetes (T2D) and, more recently, cancer. However, the conservation of binding sites between the liver and muscle isoforms makes the inhibitor selectivity challenging. Using a combination of kinetic, crystallographic, modeling, and cellular studies, we have probed the binding of dietary flavonoids epigallocatechin gallate (EGCG) and epigallocatechin (EGC) to GP isoforms. The structures of rmGPb-EGCG and rmGPb-EGC complexes were determined by X-ray crystallography, showing binding at the quercetin binding site (QBS) in agreement with kinetic studies that revealed both compounds as noncompetitive inhibitors of GP, with EGCG also causing a significant reduction in cell viability and migration of U87-MG glioblastoma cells. Interestingly, EGCG exhibits different binding modes to GP isoforms, revealing QBS as a promising site for GP targeting, offering new opportunities for the design of liver-selective GP inhibitors.

Inhibition of glycogen phosphorylase (Pyg) - a regulatory enzyme of glycogen phosphorolysis - influences memory formation in rodents. We have previously shown that 2-week intraperitoneal administration of a Pyg inhibitor BAY U6751 stimulated the "rejuvenation" of the hippocampal proteome and dendritic spines morphology and improved cognitive skills of old mice. Given the tedious nature of daily intraperitoneal drug administration, in this study we investigated whether a single dose of BAY U6751 could induce enduring behavioral effects. Obtained results support the efficacy of such treatment in significantly improving the cognitive performance of 20-22-month-old mice. Metabolomic analysis of alterations observed in the hippocampus, cerebellum, and cortex reveal that the inhibition of glycogen phosphorolysis impacts not only glucose metabolism but also various other metabolic processes., (© 2024. The Author(s).)

Compound 1 was previously identified by our team as a glycogen phosphorylase (GP) inhibitor with glucose-lowering activity and demonstrated to have protective effects against myocardial and cerebral ischemia. However, its impact on muscle function has not been clarified. This study is the first to evaluate the long-term effects of GP inhibitors on muscle function and metabolism. After a 28-day administration of Compound 1 , we performed assays to assess muscle function and biochemical parameters in rats. We observed reductions in peak holding force, duration, tetanic contraction force, single-contraction force, and electromyographic signals under 20 s and 10 min contraction stimuli. The metabolic analysis showed no significant effects on muscle glycogen, ATP, lactic acid, and uric acid levels at low doses. In contrast, medium to high doses resulted in increased glycogen, decreased ATP, and reduced lactic acid (only at high doses), without affecting uric acid. These findings suggest that Compound 1 may adversely affect muscle function in rats, potentially due to the glycogen inhibition effects of GP inhibitors. This study provides crucial safety data and insights into the long-term effects of GP inhibitors on rat muscles, which will guide future developments and applications.

Recently studied N -(β-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 -(β-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 β-d-glucopyranosylamine and the corresponding arylimidazole-carboxylic acids. Kinetics experiments on rabbit muscle GP b revealed low micromolar inhibitors, with the best inhibition constants ( K i s) of ~3-4 µM obtained for 1- and 2-naphthyl-substituted N -(β-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.

Glycogen phosphorylase (GP) is a key regulator of glucose levels and, with that, an important target for the discovery of novel treatments against type 2 diabetes. β-d-Glucopyranosyl derivatives have provided some of the most potent GP inhibitors discovered to date. In this regard, C -β-d-glucopyranosyl azole type inhibitors proved to be particularly effective, with 2- and 4-β-d-glucopyranosyl imidazoles among the most potent designed to date. His377 backbone C=O hydrogen bonding and ion-ion interactions of the protonated imidazole with Asp283 from the 280s loop, stabilizing the inactive state, were proposed as crucial to the observed potencies. Towards further exploring these features, 4-amino-3-(β-d-glucopyranosyl)-5-phenyl-1 H -pyrazole ( 3 ) and 3-(β-d-glucopyranosyl)-4-guanidino-5-phenyl-1 H -pyrazole ( 4 ) were designed and synthesized with the potential to exploit similar interactions. Binding assay experiments against rabbit muscle GPb revealed 3 as a moderate inhibitor (IC 50 = 565 µM), but 4 displayed no inhibition at 625 µM concentration. Towards understanding the observed inhibitions, docking and post-docking molecular mechanics-generalized Born surface area (MM-GBSA) binding free energy calculations were performed, together with Monte Carlo and density functional theory (DFT) calculations on the free unbound ligands. The computations revealed that while 3 was predicted to hydrogen bond with His377 C=O in its favoured tautomeric state, the interactions with Asp283 were not direct and there were no ion-ion interactions; for 4 , the most stable tautomer did not have the His377 backbone C=O interaction and while ion-ion interactions and direct hydrogen bonding with Asp283 were predicted, the conformational strain and entropy loss of the ligand in the bound state was significant. The importance of consideration of tautomeric states and ligand strain for glucose analogues in the confined space of the catalytic site with the 280s loop in the closed position was highlighted.

Objective: In the present study, we determined whether the glycogen phosphorylase(GP)inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) ameliorates pentylenetetrazole (PTZ)-induced acute seizure, neuroinflammation and memory impairment in rats., Methods: In experiment 1, rats were randomly divided into the Vehicle ( n =5) and PTZ ( n =5) groups, and received intraperitoneal injection of saline or PTZ (70 mg/kg), respectively. Hippocampal tissues were collected 30 min after drug injection. Western blot was used to examine the levels of GP expression. Colorimetric assay was used to determine the levels of lactate. In experiment 2, rats were randomly divided into the Vehicle+Vehicle ( n =18), DAB+Vehicle ( n =18), Vehicle+PTZ ( n =19) and DAB+PTZ ( n =18) groups. Rats received intracerebroventricular injection of PBS or DAB (50 μg/2 μl) 15 min before receiving intraperitoneal injection of saline or PTZ (70 mg/kg). Behavioural assays and the Racine scale were used to evaluate seizure severity. Western blot was used to examine the levels of targeted protein of hippocampal tissues. Novel object recognition test was used to assess memory performance., Results: ① Compared with the Vehicle group, the levels of GP and lactate in the hippocampal tissues of the PTZ group were increased significantly (both P ＜0.01). ② Compared with the Vehicle+PTZ group, in the DAB+PTZ group, the levels of myoclonic body jerk latency, forelimb clonus latency and tonic-clonic seizure latency were increased significantly (all P ＜0.01), while the duration of seizure and seizure scores were decreased significantly (both P ＜0.01). ③ Compared with the Vehicle+Vehicle group, in the Vehicle +PTZ group, the levels of IL-1β, IL-6, TNF-α, IBA-1 and GFAP in the hippocampal tissues were increased significantly (all P ＜0.01), and the discrimination index in the novel object recognition test was decreased significantly ( P ＜0.01). Compared with the Vehicle+PTZ group, in the DAB+PTZ group, the levels of IL-1β, TNF-α, IBA-1 and GFAP in the hippocampal tissues were decreased significantly (all, P ＜0.01), while the discrimination index in the novel object recognition test was increased significantly ( P ＜0.01)., Conclusion: DAB ameliorates PTZ-induced seizure, neuroinflammation and memory impairment in rats, suggesting that DAB may serve as a novel agent for potential clinical treatment of epilepsy.

Molecular rotors belong to a family of fluorescent compounds characterized as molecular switches, where a fluorescence on/off signal signifies a change in the molecule's microenvironment. Herein, the successful synthesis and detailed study of ( E )-2-cyano-3-( p -(dimethylamino)phenyl)- N -(β-D-glucopyranosyl)acrylamide (RotA), is reported. RotA was found to be a strong inhibitor of rabbit muscle glycogen phosphorylase (RMGP b ), that binds at the catalytic site of the enzyme. RotA's interactions with the residues lining the catalytic site of RMGP b were determined by X-ray crystallography. Spectroscopic studies coupled with theoretical calculations proved that RotA is a molecular rotor. When bound in the catalytic channel of RMGP b , it behaved as a light switch, generating a strong fluorescence signal, allowing utilization of RotA as a probe that locates glycogen phosphorylase (GP). RotA, mono-, di- and per-acetylated derivatives, as well as nanoparticles with RotA encapsulated in polyethylene glycol-poly-L-histidine, were used in live cell fluorescence microscopy imaging to test the delivery of RotA through the plasma membrane of HepG2 and A431 cells, with the nanoparticles providing the best results. Once in the intracellular milieu, RotA exhibits remarkable colocalization with GP and significant biological effects, both in cell growth and inhibition of GP.

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway. GP inhibitors are currently under investigation as a new liver-targeted approach to managing type 2 diabetes mellitus (DM). The aim of the present study was to evaluate the inhibitory activity of a panel of 52 structurally related chromone derivatives; namely, flavonoids, 2-styrylchromones, 2-styrylchromone-related derivatives [2-(4-arylbuta-1,3-dien-1-yl)chromones], and 4- and 5-styrylpyrazoles against GP, using in silico and in vitro microanalysis screening systems. Several of the tested compounds showed a potent inhibitory effect. The structure-activity relationship study indicated that for 2-styrylchromones and 2-styrylchromone-related derivatives, the hydroxylations at the A and B rings, and in the flavonoid family, as well as the hydroxylation of the A ring, were determinants for the inhibitory activity. To support the in vitro experimental findings, molecular docking studies were performed, revealing clear hydrogen bonding patterns that favored the inhibitory effects of flavonoids, 2-styrylchromones, and 2-styrylchromone-related derivatives. Interestingly, the potency of the most active compounds increased almost four-fold when the concentration of glucose increased, presenting an IC 50 < 10 µM. This effect may reduce the risk of hypoglycemia, a commonly reported side effect of antidiabetic agents. This work contributes with important considerations and provides a better understanding of potential scaffolds for the study of novel GP inhibitors.

Repeated early environmental deprivation is regarded as a typical paradigm to mimic the behavioral abnormalities and brain dysfunction that occur in psychiatric disorders. Previously, we reported that social isolation could disrupt prepulse inhibition (PPI) in Sprague-Dawley (SD) rats, producing the typical characteristics of a schizophrenia animal model. Based on further analysis of previous proteomic and transcriptomic data, a disrupted balance of glucose metabolism was found in the prefrontal cortex (PFC) of isolated rats. Subsequently, in the first experiment of this study, we investigated the effects of juvenile social isolation (postnatal days (PND) 21-34) on PPI and lactate levels in PND56 rats. Compared with the social rearing group, rats in the isolated rearing group showed disrupted PPI and increased lactate levels in the PFC. In the second experiment, at PND55, the model rats were acutely injected with a glycogen phosphorylase inhibitor (4-dideoxy-1,4-imino-darabinitol, DAB) or control saline in the bilateral PFC. Our data showed that acute DAB administration (50 pmol, 0.5 μl) significantly improved the disrupted PPI and decreased the levels of oxidative phosphorylation (OXPHOS)-related mRNAs as well as lactate. In summary, our results suggested that excess astrocytic lactate production was involved in the impairment of auditory sensory gating of isolated rats, which may contribute to the metabolic pathogenesis of schizophrenia., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 IC 50 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.

The purine alkaloid caffeine is the most widely consumed psychostimulant drug in the world and has multiple beneficial pharmacological activities, for example, in neurodegenerative diseases. However, despite being an extensively studied bioactive natural product, the mechanistic understanding of caffeine's pharmacological effects is incomplete. While several molecular targets of caffeine such as adenosine receptors and phosphodiesterases have been known for decades and inspired numerous medicinal chemistry programs, new protein interactions of the xanthine are continuously discovered providing potentially improved pharmacological understanding and a molecular basis for future medicinal chemistry. In this Perspective, we gather knowledge on the confirmed protein interactions, structure activity relationship, and chemical biology of caffeine on well-known and upcoming targets. The diversity of caffeine's molecular activities on receptors and enzymes, many of which are abundant in the CNS, indicates a complex interplay of several mechanisms contributing to neuroprotective effects and highlights new targets as attractive subjects for drug discovery.

Glycogen phosphorylase (GP) is a key enzyme of glycogen catabolism, so it is significant to discover a new GP inhibitor. A series of benzazepinone derivatives were discovered as GP inhibitors with potent activity. Among these derivatives, compound 5d showed significant potential against rabbit muscle GPa (IC 50  = 0.25 ± 0.05 μM) and cellular efficacy. The in vivo study revealed that 5d significantly inhibited increases in fasting blood glucose level in two kinds of hyperglycemic mice models. The possible binding mode of compound 5d was explored based on molecular docking simulations. These results indicated that derivatives with benzazepinone were potential chemical entities against hyperglycemia.

Novel pharmacological strategies for the treatment of diabetic patients are now focusing on inhibiting glycogenolysis steps. In this regard, glycogen phosphorylase (GP) is a validated target for the discovery of innovative antihyperglycemic molecules. Natural products, and in particular flavonoids, have been reported as potent inhibitors of GP at the cellular level. Herein, free-energy calculations and microscale thermophoresis approaches were performed to get an in-depth assessment of the binding affinities and elucidate intermolecular interactions of several flavonoids at the inhibitor site of GP. To our knowledge, this is the first study indicating genistein, 8-prenylgenistein, apigenin, 8-prenylapigenin, 8-prenylnaringenin, galangin and valoneic acid dilactone as natural molecules with high inhibitory potency toward GP. We identified: i) the residues Phe285, Tyr613, Glu382 and/or Arg770 as the most relevant for the binding of the best flavonoids to the inhibitor site of GP, and ii) the 5-OH, 7-OH, 8-prenyl substitutions in ring A and the 4'-OH insertion in ring B to favor flavonoid binding at this site. Our results are invaluable to plan further structural modifications through organic synthesis approaches and develop more effective pharmaceuticals for Type 2 Diabetes treatment, and serve as the starting point for the exploration of food products for therapeutic usage, as well as for the development of novel bio-functional food and dietary supplements/herbal medicines., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Type 2 diabetes mellitus (T2DM), one of the most common metabolic diseases, is characterized by insulin resistance and inadequate insulin secretion of β cells. Glycogen phosphorylase (GP) is the key enzyme in glycogen breakdown, and contributes to hepatic glucose production during fasting or during insulin resistance. Pharmacological GP inhibitors are potential glucose lowering agents, which may be used in T2DM therapy. A natural product isolated from the cultured broth of the fungal strain No. 138354, called 2,3-bis(4-hydroxycinnamoyloxy)glutaric acid (FR258900), was discovered a decade ago. In vivo studies showed that FR258900 significantly reduced blood glucose levels in diabetic mice. We previously showed that GP inhibitors can potently enhance the function of β cells. The purpose of this study was to assess whether an analogue of FR258900 can influence β cell function. BF142 (Meso-Dimethyl 2,3-bis[(E)-3-(4-acetoxyphenyl)prop-2-enamido]butanedioate) treatment activated the glucose-stimulated insulin secretion pathway, as indicated by enhanced glycolysis, increased mitochondrial oxidation, significantly increased ATP production, and elevated calcium influx in MIN6 cells. Furthermore, BF142 induced mTORC1-specific phosphorylation of S6K, increased levels of PDX1 and insulin protein, and increased insulin secretion. Our data suggest that BF142 can influence β cell function and can support the insulin producing ability of β cells., Competing Interests: The authors have declared that no competing interests exist.

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.

Glycogen phosphorylase (GP) is an important target for the development of new anti-hyperglycaemic agents. Flavonoids are novel inhibitors of GP, but their mode of action is unspecific in terms of the GP binding sites involved. Towards design of synthetic flavonoid analogues acting specifically at the inhibitor site and to exploit the site's hydrophobic pocket, chrysin has been employed as a lead compound for the in silico screening of 1169 new analogues with different B ring substitutions. QM/MM-PBSA binding free energy calculations guided the final selection of eight compounds, subsequently synthesised using a Baker-Venkataraman rearrangement-cyclisation approach. Kinetics experiments against rabbit muscle GPa and GPb together with human liver GPa, revealed three of these compounds (11, 20 and 43) among the most potent that bind at the site (K i s < 4 µM for all three isoforms), and more potent than previously reported natural flavonoid inhibitors. Multiple inhibition studies revealed binding exclusively at the inhibitor site. The binding is synergistic with glucose suggesting that inhibition could be regulated by blood glucose levels and would decrease as normoglycaemia is achieved. Compound 43 was an effective inhibitor of glycogenolysis in hepatocytes (IC 50  = 70 µM), further promoting these compounds for optimization of their drug-like potential. X-ray crystallography studies revealed the B-ring interactions responsible for the observed potencies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Brain-type glycogen phosphorylase (pygb) is one of the rate-limiting enzymes in glycogenolysis that plays a crucial role in the pathogenesis of type 2 diabetes mellitus. Here we investigated the role of pygb in high-glucose (HG)-induced cardiomyocyte apoptosis and explored the underlying mechanisms, by using the specific pygb inhibitors or pygb siRNA. Our results show that inhibition of pygb significantly attenuates cell apoptosis and oxidative stress induced by HG in H9c2 cardiomyocytes. Inhibition of pygb improved glucose metabolism in cardiacmyocytes, as evidenced by increased glycogen content, glucose consumption, and glucose transport. Mechanistically, pygb inhibition activates the Akt-GSK-3β signaling pathway and suppresses the activation of NF-κB in H9c2 cells exposed to HG. Additionally, pygb inhibition promotes the expression and the translocation of hypoxia-inducible factor-1α (HIF-1α) after HG stimulation. However, the changes in glucose metabolism and HIF-1α activation mediated by pygb inhibition are significantly reversed in the presence of the Akt inhibitor MK2206. In conclusion, this study found that inhibition of pygb prevents HG-induced cardiomyocyte apoptosis via activation of Akt-HIF-α.

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 (IC 50  = 0.29 μM), good cellular efficacy (IC 50  = 3.24 μM for HepG2 cells and IC 50  = 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Brain glycogen is a vital energy source during metabolic imbalance. Metabolic sensory neurons in the ventromedial hypothalamic nucleus (VMN) shape glucose counter-regulation. Insulin-induced hypoglycemic (IIH) male rats were infused icv with the glycogen breakdown inhibitor CP-316,819 (CP) to investigate whether glycogen-derived fuel controls basal and/or hypoglycemic patterns of VMN gluco-regulatory neuron energy stability and transmitter signaling. CP caused dose-dependent amplification of basal VMN glycogen content and either mobilization (low dose) or augmentation (high dose) of this depot during IIH. Drug treatment also prevented hypoglycemic diminution of tissue glucose in multiple structures. Low CP dose caused IIH-reversible augmentation of AMPK activity and glutamate decarboxylase (GAD) protein levels in laser-microdissected VMN GABA neurons, while the higher dose abolished hypoglycemic adjustments in these profiles. VMN steroidogenic factor-1 (SF-1) neurons exhibited suppressed (low CP dose) or unchanged (high CP dose) basal SF-1 expression and AMPK refractoriness of hypoglycemia at each dose. CP caused dose-proportionate augmentation of neuronal nitric oxide synthase protein and enhancement (low dose) or diminution (high dose) of this profile during IIH; AMPK activity in these cells was decreased in high dose-pretreated IIH rats. CP exerted dose-dependent effects on basal and hypoglycemic patterns of glucagon, but not corticosterone secretion. Results verify that VMN GABA, SF-1, and nitrergic neurons are metabolic sensory in function and infer that these populations may screen unique aspects of neurometabolic instability. Correlation of VMN glycogen augmentation with attenuated hypoglycemic VMN gluco-regulatory neuron AMPK activity implies that expansion of this fuel reservoir preserves cellular energy stability during this metabolic threat.

The design of glycogen phosphorylase (GP) inhibitors targeting the catalytic site of the enzyme is a promising strategy for a better control of hyperglycaemia in the context of type 2 diabetes. Glucopyranosylidene-spiro-heterocycles have been demonstrated as potent GP inhibitors, and more specifically spiro-oxathiazoles. A new synthetic route has now been elaborated through 1,3-dipolar cycloaddition of an aryl nitrile oxide to a glucono-thionolactone affording in one step the spiro-oxathiazole moiety. The thionolactone was obtained from the thermal rearrangement of a thiosulfinate precursor according to Fairbanks' protocols, although with a revisited outcome and also rationalised with DFT calculations. The 2-naphthyl substituted glucose-based spiro-oxathiazole 5h, identified as one of the most potent GP inhibitors (K i = 160 nM against RMGPb) could be produced on the gram-scale from this strategy. Further evaluation in vitro using rat and human hepatocytes demonstrated that compound 5h is a anti-hyperglycaemic drug candidates performing slightly better than DAB used as a positive control. Investigation in Zucker fa/fa rat model in acute and subchronic assays further confirmed the potency of compound 5h since it lowered blood glucose levels by ∼36% at 30 mg kg -1 and ∼43% at 60 mg kg -1 . The present study is one of the few in vivo investigations for glucose-based GP inhibitors and provides data in animal models for such drug candidates.

Despite the substantial interest in C -glycosyl heterocycles as mimetics of biologically active native glycans, the appearance of C -glycopyranosyl derivatives of six-membered heterocycles, both in synthetic and biological contexts, is rather scarce. As part of our ongoing research program aimed at preparing hitherto barely known 2- C -glycopyranosyl pyrimidines, the goal of the present study was to synthesize new 5-mono- and multiply substituted derivatives of this compound class. Thus, 2- C -(β-D-glucopyranosyl)-5,6-disubstituted-pyrimidin-4(3 H )-ones and 4-amino-2- C -(β-D-glucopyranosyl)-5,6-disubstituted-pyrimidines were prepared by base-mediated cyclocondensations of O -perbenzylated and O -unprotected C -(β-D-glucopyranosyl) formamidine hydrochlorides with methylenemalonic acid derivatives. The 2- C -(β-D-glucopyranosyl)-5-substituted-pyrimidines were obtained from the same amidine precursors upon treatment with vinamidinium salts. The deprotected derivatives of these pyrimidines were tested as inhibitors of some glycoenzymes. None of them showed inhibitory activity towards glycogen phosphorylase and α- and β-glucosidase enzymes, but some members of the sets exhibited moderate inhibition against bovine liver β-galactosidase., Competing Interests: The authors declare no conflict of interest.

In this study, we report the ring transformation of 3-arylsydnone into 1-aryl-1H-pyrazole-3-carbonitriles via [3 + 2] cycloaddition with acrylonitrile. 1-Aryl-1H-pyrazole-3-carbonitrile underwent [2 + 3] cycloaddition with sodium azide to afford 5-(1-aryl-1H-pyrazol-3-yl)-1H-tetrazoles which were further subjected to N-alkylation with aryl/heteroaryl alkyl halides to afford 1,5- and 2,5-disubstituted tetrazoles. Furthermore, the title compounds were screened for in vivo antihyperglycemic activity using albino Wistar rats of either sex. Compounds 4a, 6b, 7a, 7b, 8b, and 9b showed maximum fall in the blood glucose levels in streptozotocin-induced diabetic rats after 5-7 days of administration. In support of antidiabetic activity, we also performed the experimental in vivo studies, namely, effect of compounds on enzymes (serum glutamic oxaloacetic transaminase, serum glutamic-pyruvic transaminase, creatinine, urea, and total protein), antihyperlipidemic, and histopathology. Moreover, the molecular docking study has been performed for potent molecules among the series with glycogen phosphorylase as target enzyme, and this study corroborated the experimental in vivo results., (© 2019 Wiley Periodicals, Inc.)

To verify the effect of protein phosphorylation on glycolysis and elucidate the regulatory mechanism from the perspective of enzyme activity, ovine muscle was treated with a kinase inhibitor, dimethyl sulfoxide, or a phosphatase inhibitor and the activities of glycogen phosphorylase, pyruvate kinase and phosphofructokinase were determined. The protein phosphorylation level was significantly different after incubation of muscle with kinase or phosphatase inhibitors. The pH value and lactate content revealed that a high phosphorylation level was the reason for the fast glycolysis. The glycogen phosphorylase, pyruvate kinase and phosphofructokinase activities were significantly higher in the phosphatase inhibitor group than in the other two groups (p < 0.05). Therefore, protein phosphorylation is involved in activating these three enzymes. In summary, protein phosphorylation plays a role in post-mortem glycolysis through the regulation of enzyme activity in ovine muscle., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hepatocellular carcinoma (HCC) is one of the leading cancers in the world in incidence and mortality. Current pharmacotherapy of HCC is limited in the number and efficacy of anticancer agents. Metabolic reprogramming is a prominent feature of many cancers and has rekindled interest in targeting metabolic proteins for cancer therapy. Glycogen is a storage form of glucose, and the levels of glycogen have been found to correlate with biological processes in reprogrammed cancer cells. However, the contribution of glycogen metabolism to carcinogenesis, cancer cell growth, metastasis, and chemoresistance is poorly understood. Thus, we studied the processes involved in the inhibition of glycogen metabolism in HCC cells. Pharmacological inhibition of glycogen phosphorylase (GP), a rate-limiting enzyme in glycogen catabolism, by CP-91149 led to a decrease in HCC cell viability. GP inhibition induced cancer cell death through the intrinsic apoptotic pathway. Mitochondrial dysfunction and autophagic adaptations accompanied this apoptosis process whereas endoplasmic reticulum stress, necrosis, and necroptosis were not major components of the cell death. In addition, GP inhibition potentiated the effects of multikinase inhibitors sorafenib and regorafenib, which are key drugs in advanced-stage HCC therapy. Our study provides mechanistic insights into cell death by perturbation of glycogen metabolism and identifies GP inhibition as a potential HCC pharmacotherapy target., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Several C -β-d-glucopyranosyl azoles have recently been uncovered as among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Toward further exploring their translational potential, ex vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the molecular mechanics-generalized Born surface area (MM-GBSA) method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson ( R P ) and Spearman ( R S ) correlations (both 0.98), predictive index (PI = 0.99), and area under the receiver operating characteristic curve (AU-ROC = 0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using density functional theory (DFT). Seven 2-aryl-4(5)-(β-d-glucopyranosyl)-imidazoles and 2-aryl-4-(β-d-glucopyranosyl)-thiazoles were subsequently synthesized, and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best K i values in the low micromolar range ( 5c = 1.97 μM; 13b = 4.58 μM). Ten C -β-d-glucopyranosyl azoles were then tested ex vivo in mouse primary hepatocytes. Four of these ( 5a - c and 9d ) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC 50 = 30-60 μM). Structural and predicted physicochemical properties associated with their effectiveness were analyzed with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring, and the calculated p K a (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favored, while within the cell, there is predicted more favorable binding to GP in the protonated form.

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 ( K i 1.7 μM) and also acted on HepG2 cells (IC 50 177 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.

In the case of type 2 diabetes, inhibitors of glycogen phosphorylase (GP) may prevent unwanted glycogenolysis under high glucose conditions and thus aim at the reduction of excessive glucose production by the liver. Anomeric spironucleosides, such as hydantocidin, present a rich synthetic chemistry and important biological function (e.g., inhibition of GP). For this study, the Suárez radical methodology was successfully applied to synthesize the first example of a 1,6-dioxa-4-azaspiro[4.5]decane system, not previously constructed via a radical pathway, starting from 6-hydroxymethyl-β-d-glucopyranosyluracil. It was shown that, in the rigid pyranosyl conformation, the required [1,5]-radical translocation was a minor process. The stereochemistry of the spirocycles obtained was unequivocally determined based on the chemical shifts of key sugar protons in the 1 H-NMR spectra. The two spirocycles were found to be modest inhibitors of RMGP b .

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 PPh 3 . Catalytic hydrogenation (H 2 -Pd/C) or partial reduction (e.g., H 2 -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).

This review is an effort to summarize recent developments in synthesis of O-glycosides and N-, C-glycosyl molecules with promising antidiabetic potential. Articles published after 2000 are included. First, the O-glycosides used in the treatment of diabetes are presented, followed by the N-glycosides and finally the C-glycosides constituting the largest group of antidiabetic drugs are described. Within each group of glycosides, we presented how the structure of compounds representing potential drugs changes and when discussing chemical compounds of a similar structure, achievements are presented in the chronological order. C-Glycosyl compounds mimicking O-glycosides structure, exhibit the best features in terms of pharmacodynamics and pharmacokinetics. Therefore, the largest part of the article is concerned with the description of the synthesis and biological studies of various C-glycosides. Also N-glycosides such as N-(β-D-glucopyranosyl)-amides, N-(β-D-glucopyranosyl)-ureas, and 1,2,3-triazolyl derivatives belong to the most potent classes of antidiabetic agents. In order to indicate which of the compounds presented in the given sections have the best inhibitory properties, a list of the best inhibitors is presented at the end of each section. In summary, the best inhibitors were selected from each of the summarizing figures and the results of the ranking were placed. In this way, the reader can learn about the structure of the compounds having the best antidiabetic activity. The compounds, whose synthesis was described in the article but did not appear on the figures presenting the structures of the most active inhibitors, did not show proper activity as inhibitors. Thus, the article also presents studies that have not yielded the desired results and show directions of research that should not be followed. In order to show the directions of the latest research, articles from 2018 to 2019 are described in a separate Sect. 5. In Sect. 6, biological mechanisms of action of the glycosides and patents of marketed drugs are described.

A fluorescence study of N1-(β-d-glucopyranosyl)-N4-[2-acridin-9(10H)-onyl]-cytosine (GLAC), the first fluorescent potent inhibitor of glycogen phosphorylase (GP), in neutral aqueous solution, is presented herein. Quantum chemistry (TD-DFT) calculations show the existence of several conformers both in the ground and first excited states. They result from rotations of the acridone and cytosine moieties around an NH bridge which may lead to the formation of non-emitting charge-transfer states. The fingerprints of various conformers have been detected by time-resolved fluorescence spectroscopy (fluorescence upconversion and time-correlated single photon counting) and identified using as criteria their energy, polarization and relative population resulting from computations. Such an analysis should contribute to the design of new GP inhibitors with better fluorescence properties, suitable for imaging applications.

Structure-based design and synthesis of two biphenyl- N -acyl-β-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.

Acanthamoeba keratitis is an ophthalmic disease with no specific treatment that specially affects contact lens users. The silencing of serine phosphatase (SP) and glycogen phosphorylase (GP) proteins produced by Acanthamoeba has been shown to significantly reduce the cytopathic effect, although no vehicle was proposed yet to deliver the siRNA sequences to the trophozoites. In this study, PEGylated cationic liposomes were proposed and optimized using Box-Behnken design. The influence of DOTAP:DOPE ratio, DSPE-PEG concentration, and siRNA/DOTAP charge ratio were evaluated over both biological response and physicochemical properties of liposomes. The ratio of DOTAP:DOPE had an effect in the trophozoite activity whereas the charge ratio influenced both size and protease activity. The predicted values were very close to the observed values, yielding a formulation with good activity and toxicity profile, which was used in the following experiments. A murine model of ocular keratitis was treated with siGP + siSP-loaded liposomes, as well as their respective controls, and combined treatment of liposomes and chlorhexidine. After 15 days of eight daily administrations, the liposomal complex combined with chlorhexidine was the only treatment able to reverse the more severe lesions associated with keratitis. There was 60% complete regression in corneal damage, with histological sections demonstrating the presence of an integral epithelium, without lymphocytic infiltrate. The set of results demonstrate the efficacy of a combined therapy based on siRNA with classical drugs for a better prognosis of keratitis caused by Acanthamoeba., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A series of pentacyclic triterpenoids similar to glycyrrhetinic acid were designed and synthesized via the combination of chemical modification and microbial catalysis. All products were screened for the glycogen phosphorylases inhibitory activities in vitro. Within this series of derivatives, compound 5 displayed good inhibitory activities with IC 50 value of 27.7 μM, which is better than that of the other derivatives and glycyrrhetinic acid. Structure-activity relationship (SAR) analysis of these inhibitors was also discussed.

In the field of immunology, there is an increasing interest in cellular energy metabolism and its outcome on immune cell effector function. Activation of immune cells leads to rapid metabolic changes that are central to cellular biology in order to support the effector responses. Therefore, the need for user-friendly and dependable assay technologies to address metabolic regulation and nutrient utilization in immune cells is an important need in this field. Redox-dye reduction-based Phenotype MicroArray (PM) assays, which measure NADH reduction as a readout, developed by Biolog Inc., provide a wide screening of metabolites both in bacteria and mammalian cells. In this study, we delineate a detailed protocol of a customized Biolog assay for investigation of a specific metabolic pathway of interest. The option to be able to easily customize this technology offers researchers with a convenient assay platform to methodically examine specific nutrient substrates or metabolic pathways of interest in a rapid and cost effective manner., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A few new anthranilate diamide derivatives, 3a ⁻ e , 5a ⁻ c and 7a ⁻ 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; 13 C 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 IC 50 = 0.01247 ± 0.01 µM and IC 50 = 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.

Human liver glycogen phosphorylase (hlGP), a key enzyme in glycogen metabolism, is a valid pharmaceutical target for the development of new anti-hyperglycaemic agents for type 2 diabetes. Inhibitor discovery studies have focused on the active site and in particular on glucopyranose based compounds with a β-1 substituent long enough to exploit interactions with a cavity adjacent to the active site, termed the β-pocket. Recently, C-β-d-glucopyranosyl imidazoles and 1, 2, 4-triazoles proved to be the best known glucose derived inhibitors of hlGP. Here we probe the β-pocket by studying the inhibitory effect of six different groups at the para position of 3-(β-d-glucopyranosyl phenyl)-5-phenyl-, 1, 2, 4-triazoles in hlGP by kinetics and X-ray crystallography. The most bioactive compound was the one with an amine substituent to show a K i value of 0.43 μM. Structural studies have revealed the physicochemical diversity of the β-pocket providing information for future rational inhibitor design studies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

3-(β-d-Glucopyranosyl)-5-substituted-1,2,4-triazoles have been revealed as an effective scaffold for the development of potent glycogen phosphorylase (GP) inhibitors but with the potency very sensitive to the nature of the alkyl/aryl 5-substituent (Kun et al., Eur. J. Med. Chem. 2014, 76, 567). For a training set of these ligands, quantum mechanics-polarized ligand docking (QM-PLD) demonstrated good potential to identify larger differences in potencies (predictive index PI = 0.82) and potent inhibitors with K i 's < 10 μM (AU-ROC = 0.86). Accordingly, in silico screening of 2335 new analogues exploiting the ZINC docking database was performed and nine predicted candidates selected for synthesis. The compounds were prepared in O-perbenzoylated forms by either ring transformation of 5-β-d-glucopyranosyl tetrazole by N-benzyl-arenecarboximidoyl chlorides, ring closure of C-(β-d-glucopyranosyl)formamidrazone with aroyl chlorides, or that of N-(β-d-glucopyranosylcarbonyl)arenethiocarboxamides by hydrazine, followed by deprotections. Kinetics experiments against rabbit muscle GPb (rmGPb) and human liver GPa (hlGPa) revealed five compounds as potent low μM inhibitors with three of these on the submicromolar range for rmGPa. X-ray crystallographic analysis sourced the potency to a combination of favorable interactions from the 1,2,4-triazole and suitable aryl substituents in the GP catalytic site. The compounds also revealed promising calculated pharmacokinetic profiles., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background and Purpose: Glycogen phosphorylase (GP) is the key enzyme for glycogen degradation. GP inhibitors (GPi-s) are glucose lowering agents that cause the accumulation of glucose in the liver as glycogen. Glycogen metabolism has implications in beta cell function. Glycogen degradation can maintain cellular glucose levels, which feeds into catabolism to maintain insulin secretion, and elevated glycogen degradation levels contribute to glucotoxicity. The purpose of this study was to assess whether influencing glycogen metabolism in beta cells by GPi-s affects the function of these cells., Experimental Approach: The effects of structurally different GPi-s were investigated on MIN6 insulinoma cells and in a mouse model of diabetes., Key Results: GPi treatment increased glycogen content and, consequently, the surface area of glycogen in MIN6 cells. Furthermore, GPi treatment induced insulin receptor β (InsRβ), Akt and p70S6K phosphorylation, as well as pancreatic and duodenal homeobox 1(PDX1) and insulin expression. In line with these findings, GPi-s enhanced non-stimulated and glucose-stimulated insulin secretion in MIN6 cells. The InsRβ was shown to co-localize with glycogen particles as confirmed by in silico screening, where components of InsR signalling were identified as glycogen-bound proteins. GPi-s also activated the pathway of insulin secretion, indicated by enhanced glycolysis, mitochondrial oxidation and calcium signalling. Finally, GPi-s increased the size of islets of Langerhans and improved glucose-induced insulin release in mice., Conclusion and Implications: These data suggest that GPi-s also target beta cells and can be repurposed as agents to preserve beta cell function or even ameliorate beta cell dysfunction in different forms of diabetes., Linked Articles: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc., (© 2017 The British Pharmacological Society.)

Prevention of the formation of advanced glycation end-products (AGEs) is a reliable approach to achieve control over hyperglycemia and the associated pathogenesis of diabetic vascular complications. In these terms, new synthetic approaches to 6-nitroazolo[1,5-a]pyrimidines have been developed on the basis of the promising antiglycation activity of their structural analogues, such as azolo[5,1-c][1,2,4]triazine-4(1H)-ones. A number of nitroazolopyrimidines were obtained by using nitration, chlorodeoxygenation, and amination reactions, and their antidiabetic properties were elucidated in vitro. It was shown that triazolo[1,5-a]pyrimidine-7(4H)-ones exhibit a higher antiglycation activity than the corresponding 7-alkylamino analogs and aminoguanidine, as the reference compound. It is suggested that this kind of activity can be associated with the chelating properties possessed by the synthesized 6-nitro-7-oxoderivatives. Furthermore, the compounds obtained were tested for their inhibitory activity against dipeptidyl peptidase 4 (DPP4), glycogen phosphorylase, and α-glucosidase in vitro, but their activities proved to be significantly inferior to those of the reference compounds., (© 2017 Deutsche Pharmazeutische Gesellschaft.)

The glucose polymer glycogen is a vital fuel reserve in the brain. The mediobasal hypothalamic energy sensor AMP-activated protein kinase (AMPK) maintains glucostasis via neurotransmitter mechanisms that suppress [ γ -aminobutyric acid; GABA] or stimulate [nitric oxide; steroidogenic factor-1 (SF1)] counter-regulatory outflow. This study investigated whether glycogen-derived fuel supply is a critical screened variable in ventromedial hypothalamic nucleus (VMN) monitoring of neuro-metabolic stability during glucostasis and/or insulin (I)-induced hypoglycemia. Adult male rats were pretreated by intra-VMN infusion of the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) before sc vehicle or I injection. Western blot analyses of micropunch-dissected VMN tissue from euglycemic animals showed DAB augmentation of phosphoAMPK (pAMPK), neuronal nitric oxide synthase (nNOS), and SF-1, but not glutamate decarboxylase 65/67 (GAD) protein. Combinatory DAB/I treatment did not further enhance AMPK activity but significantly amplified nNOS expression relative to DAB alone. Hypoglycemic stimulation of corticosterone, but not glucagon release was prevented by DAB Results imply that glycogen-derived substrate fuel provision represses VMN AMPK activity and neurotransmitter signals of metabolic deficiency. Progressive augmentation of nNOS protein by DAB/I versus DAB/V intimates that "fuel-inhibited" nitrergic neurons may exhibit increasing sensitivity to disrupted glycogen breakdown during glucoprivation versus glucostasis. nNOS and GAD reactivity to DAB/I, but not I implies that acute glycogen utilization during hypoglycemia may be sufficiently robust to avert effects on local metabolic sensory signaling. DAB/I upregulation of GAD alongside prevention of hypercorticosteronemia suggests that indicators of metabolic sufficiency may occur secondary to local compensatory adaptations to severe restriction of glucose-derived energy., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Aryl substituted 1-(β-d-glucosaminyl)-1,2,3-triazoles as well as C-β-d-glucosaminyl 1,2,4-triazoles and imidazoles were synthesized and tested as inhibitors against muscle and liver isoforms of glycogen phosphorylase (GP). While the N-β-d-glucosaminyl 1,2,3-triazoles showed weak or no inhibition, the C-β-d-glucosaminyl derivatives had potent activity, and the best inhibitor was the 2-(β-d-glucosaminyl)-4(5)-(2-naphthyl)-imidazole with a K i value of 143 nM against human liver GPa. An X-ray crystallography study of the rabbit muscle GPb inhibitor complexes revealed structural features of the strong binding and offered an explanation for the differences in inhibitory potency between glucosyl and glucosaminyl derivatives and also for the differences between imidazole and 1,2,4-triazole analogues.

Dendritic cell (DC) activation by Toll-like receptor (TLR) agonists causes rapid glycolytic reprogramming that is required to meet the metabolic demands of their immune activation. Recent efforts in the field have identified an important role for extracellular glucose sourcing to support DC activation. However, the contributions of intracellular glucose stores to these processes have not been well characterized. We demonstrate that DCs possess intracellular glycogen stores and that cell-intrinsic glycogen metabolism supports the early effector functions of TLR-activated DCs. Inhibition of glycogenolysis significantly attenuates TLR-mediated DC maturation and impairs their ability to initiate lymphocyte activation. We further report that DCs exhibit functional compartmentalization of glucose- and glycogen-derived carbons, where these substrates preferentially contribute to distinct metabolic pathways. This work provides novel insights into nutrient homeostasis in DCs, demonstrating that differential utilization of glycogen and glucose metabolism regulates their optimal immune function., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Current treatments for Acanthamoeba keratitis are unspecific. Because of the presence of the resilient cyst form of the parasite, the infection is persistent. Silencing the key protein of cyst formation, glycogen phosphorylase, has shown potential for reducing encystment processes of the Acanthamoeba trophozoite. However, a suitable carrier to protect and deliver siRNA sequences is still needed., Dotap: DOPE:DSPE-PEG liposomes were prepared by three different techniques and used to associate a therapeutic siRNA sequence. Liposomes prepared by film hydration followed by membrane extrusion were considered the most adequate ones with average size of 250 nm and zeta potential of +45 mV, being able to associate siRNA for at least 24 hr in culture medium. siRNA-liposomes could inhibit up to 66% of the encystment process. Cell viability studies demonstrated MTT reduction capacity higher than 80% after 3 hr incubation with this formulation. After 24 hr of incubation, LDH activity ranged for both the formulations from around 4% to 40%. In vivo tolerance studies in mice showed no macroscopic alteration in the eye structures up to 24 hr after eight administrations during 1 day. Histological studies showed regular tissue architecture without any morphological alteration. Overall, these results suggest that the formulations developed are a promising new strategy for the treatment of ocular keratitis caused by Acanthamoeba spp., (© 2017 John Wiley & Sons A/S.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The design and synthesis of a glucose-based acridone derivative (GLAC), a potent inhibitor of glycogen phosphorylase (GP) are described. GLAC is the first inhibitor of glycogen phosphorylase, the electronic absorption properties of which are clearly distinguishable from those of the enzyme. This allows probing subtle interactions in the catalytic site. The GLAC absorption spectra, associated with X-ray crystallography and quantum chemistry calculations, reveal that part of the catalytic site of GP behaves as a highly basic environment in which GLAC exists as a bis-anion. This is explained by water-bridged hydrogen-bonding interactions with specific catalytic site residues., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

3-(C-Glucopyranosyl)-5aryl-1,2,4-triazoles with an aryl moiety larger than phenyl have been shown to have strong inhibitory potency (K i values in the range of upper nM) for human liver glycogen phosphorylase (hlGP), a pharmacologically relevant target for diabetes type 2. In this study we investigate in a comparative manner the inhibitory effect of the above triazoles and their respective imidazoles on hlGPa. Kinetic studies show that the imidazole derivatives are 6-8 times more potent than their corresponding triazoles. We also seek to answer how the type of the aryl moiety affects the potency in hlGPa, and by determination of the crystal structure of rmGPb in complex with the triazole derivatives the structural basis of their inhibitory efficacy is also elucidated. Our studies revealed that the van der Waals interactions between the aryl moiety and residues in a hydrophobic pocket within the active site are mainly responsible for the variations in the potency of these inhibitors., (Copyright © 2017 Elsevier Inc. All rights reserved.)

PSN-357, an effective glycogen phosphorylase (GP) inhibitor for the treatment for type 2 diabetics, is hampered in its clinical use by the poor selectivity between the GP isoforms in liver and in skeletal muscle. In this study, by the introduction of cholic acid, 9 novel potent and liver-targeted conjugates of PSN-357 were obtained. Among these conjugates, conjugate 6 exhibited slight GP inhibitory activity (IC 50  = 31.17 μM), good cellular efficacy (IC 50  = 13.39 μM) and suitable stability under various conditions. The distribution and pharmacokinetic studies revealed that conjugate 6 could redistribute from plasma to liver resulting in a considerable higher exposure of PSN-357 metabolizing from 6 in liver (AUC liver /AUC plasma ratio was 18.74) vs that of PSN-357 (AUC liver /AUC plasma ratio was 10.06). In the in vivo animal study of hypoglycemia under the same dose of 50 mg/kg, conjugate 6 exhibited a small but significant hypoglycemic effects in longer-acting manners, that the hypoglycemic effects of 6 is somewhat weaker than PSN-357 from administration up to 6 h, and then became higher than PSN-357 for the rest time of the test. Those results indicate that the liver-targeted glycogen phosphorylase inhibitor may hold utility in the treatment of type 2 diabetes.

Glycogen Phosphorylase (GP) is a functionally active dimeric enzyme, which is a target for inhibition of the conversion of glycogen to glucose-1-phosphate. In this study we report the design and synthesis of 14 new pyridone derivatives, and seek to extend the SAR analysis of these compounds. The SAR revealed the minor influence of the amide group, importance of the pyridone ring both spatially around the pyridine ring and for possible π-stacking, and confirmed a preference for inclusion of 3,4-dichlorobenzyl moieties, as bookends to the pyridone scaffold. Upon exploring a dimer strategy as part of the SAR analysis, the first extended 2-oxo-dihydropyridinyl-3-yl amide nanomolar based inhibitors of GPa (IC 50  = 230 and 260 nM) were identified., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Glycogen phosphorylase (GP) is a validated pharmaceutical target for the development of antihyperglycaemic agents. Phytogenic polyphenols, mainly flavonoids and pentacyclic triterpenes, have been found to be potent inhibitors of GP. These compounds have both pharmaceutical and nutraceutical potential for glycemic control in diabetes type 2. This review focuses mainly on the most successful (potent) of these compounds discovered to date. The protein-ligand interactions that form the structural basis of their potencies are discussed, highlighting the potential for exploitation of their scaffolds in the future design of new GP inhibitors., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)