Potent Glycogen Phosphorylase Inhibitors Research Articles

Design and Synthesis of 3-(β-d-Glucopyranosyl)-4-amino/4-guanidino Pyrazole Derivatives and Analysis of Their Glycogen Phosphorylase Inhibitory Potential.

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-1H-pyrazole (3) and 3-(β-d-glucopyranosyl)-4-guanidino-5-phenyl-1H-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 (IC50 = 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.

Discovery of novel heterocyclic derivatives as potential glycogen phosphorylase inhibitors with a cardioprotective effect

The purpose of this study was to evaluate the effect of GP inhibitor as a potential pharmaceutical target on MI/R injury. Four different structural types of novel compounds (I, II, III, and IV) were designed and synthesized, obtaining 31 novel GP inhibitors. SAR studies revealed that the conjugates of 5-chloroindole with benzo six-membered heterocyclic were found to elevate the activity. In particular, compound IIIh (IC50 = 0.21 ± 0.03 µM) emerged as a potent derivative against RMGPa, being approximately 2-fold less potent than that of PSN-357. In order to screen out a compound for in vivo activity test, we further conducted an experiment of inhibition against three different subtypes of GPa (HLGPa, HMGPa and HBGPa) and the corresponding affinity experiment. As a result, compound IIIh showed strong inhibitory activity against the above three subtypes of GP, especially on HBGPa (IC50 = 0.09 ± 0.002 µM), which was relatively close to that of positive control ingliforib (IC50 = 0.16 ± 0.02 µM). The affinity of compound IIIh to HBGPa was 4.3 times higher than that of HLGPa, and 1.1 times higher than that of HMGPa. This fact further proved that compound IIIh has a higher inhibitory effect on HMGPa than the other two subtypes. Besides, in vivo activity evaluation demonstrated that compound IIIh exhibited obviously cardioprotective effect on MI/R injury mice. The discovery of compound IIIh provides a new strategy for developing novel GP inhibitors with myocardial ischemia protection.

Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay.

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.

Noradrenaline‐induced l‐lactate production requires d‐glucose entry and transit through the glycogen shunt in single‐cultured rat astrocytes

During cognitive efforts mediated by local neuronal networks, approximately 20% of additional energy is required; this is mediated by chemical messengers such as noradrenaline (NA). NA targets astroglial aerobic glycolysis, the hallmark of which is the end product l-lactate, a fuel for neurons. Biochemical studies have revealed that astrocytes exhibit a prominent glycogen shunt, in which a portion of d-glucose molecules entering the cytoplasm is transiently incorporated into glycogen, a buffer and source of d-glucose during increased energy demand. Here, we studied single astrocytes by measuring cytosolic L-lactate ([lac]i ) with the FRET nanosensor Laconic. We examined whether NA-induced increase in [lac]i is influenced by: (a) 2-deoxy-d-glucose (2-DG, 3mM), a molecule that enters the cytosol and inhibits the glycolytic pathway; (b) 1,4-dideoxy-1,4-imino-d-arabinitol (DAB, 300µM), a potent inhibitor of glycogen phosphorylase and glycogen degradation; and (c) 3-nitropropionic acid (3-NPA, 1mM), an inhibitor of the Krebs cycle. The results of these pharmacological experiments revealed that d-glucose uptake is essential for the NA-induced increase in [lac]i , and that this exclusively arises from glycogen degradation, indicating that most, if not all, d-glucose molecules in NA-stimulated cells transit the glycogen shunt during glycolysis. Moreover, under the defined transmembrane d-glucose gradient, the glycolytic intermediates were not only used to produce l-lactate, but also to significantly support oxidative phosphorylation, as demonstrated by an elevation in [lac]i when Krebs cycle was inhibited. We conclude that l-lactate production via aerobic glycolysis is an essential energy pathway in NA-stimulated astrocytes; however, oxidative metabolism is important at rest.

Combined in silico and in vitro studies to identify novel antidiabetic flavonoids targeting glycogen phosphorylase

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.

Synthesis, Kinetic and Conformational Studies of 2-Substituted-5-(β-d-glucopyranosyl)-pyrimidin-4-ones as Potential Inhibitors of Glycogen Phosphorylase.

Dysregulation of glycogen phosphorylase, an enzyme involved in glucose homeostasis, may lead to a number of pathological states such as type 2 diabetes and cancer, making it an important molecular target for the development of new forms of pharmaceutical intervention. Based on our previous work on the design and synthesis of 4-arylamino-1-(β-d-glucopyranosyl)pyrimidin-2-ones, which inhibit the activity of glycogen phosphorylase by binding at its catalytic site, we report herein a general synthesis of 2-substituted-5-(β-d-glucopyranosyl)pyrimidin-4-ones, a related class of metabolically stable, C-glucosyl-based, analogues. The synthetic development consists of a metallated heterocycle, produced from 5-bromo-2-methylthiouracil, in addition to protected d-gluconolactone, followed by organosilane reduction. The methylthio handle allowed derivatization through hydrolysis, ammonolysis and arylamine substitution, and the new compounds were found to be potent (μM) inhibitors of rabbit muscle glycogen phosphorylase. The results were interpreted with the help of density functional theory calculations and conformational analysis and were compared with previous findings.

Anomeric Spironucleosides of β-d-Glucopyranosyl Uracil as Potential Inhibitors of Glycogen Phosphorylase.

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 1H-NMR spectra. The two spirocycles were found to be modest inhibitors of RMGPb.

Multiscale time-resolved fluorescence study of a glycogen phosphorylase inhibitor combined with quantum chemistry calculations.

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.

Immunochemical Study of the Effect of F2Glc on Glycogen Synthase Translocation and Glycogen Synthesis in Isolated Rat Hepatocytes.

The compound 2-deoxy-2-fluoro-α-D-glucopyranosyl fluoride (F2Glc), which is a nonmetabolized superior glucose analogue, is a potent inhibitor of glycogen phosphorylase and pharmacological properties are reported. Glycogen phosphorylase (GP) and glycogen synthase (GS) are responsible of the degradation and synthesis, respectively, of glycogen which is a polymer of glucose units that provides a readily available source of energy in mammals. GP and GS are two key enzymes that modulate cellular glucose and glycogen levels; therefore, these proteins are suggested as potential targets for the treatment of diseases related to glycogen metabolism disorders. We studied by Western Blot technique that F2Glc decreased GP activity, and we also showed that F2Glc did not affect GS activity and its translocation from a uniform cytosolic distribution to the hepatocyte periphery, which is crucial for glycogen synthesis, using immunoblotting and immunofluorescence labeling techniques. F2Glc specifically inhibits glycogenolysis pathway and permits a greater deposition of glycogen. These observations open up the possibility of further develop drugs that act specifically on GP. The ability to selectively inhibit GP, which is a key enzyme for the release of glucose from the hepatic glycogen reserve, may represent a new approach for the treatment of hyperglycemia in type 2 diabetes.

Frontispiece: A New Potent Inhibitor of Glycogen Phosphorylase Reveals the Basicity of the Catalytic Site

Frontispiece: A New Potent Inhibitor of Glycogen Phosphorylase Reveals the Basicity of the Catalytic Site

A New Potent Inhibitor of Glycogen Phosphorylase Reveals the Basicity of the Catalytic Site.

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.

Phytogenic Polyphenols as Glycogen Phosphorylase Inhibitors: The Potential of Triterpenes and Flavonoids for Glycaemic Control in Type 2 Diabetes.

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.

2-Oxo-1,2-dihydropyridinyl-3-yl amide-based GPa inhibitors: Design, synthesis and structure-activity relationship study

Glycogen phosphorylase (GP), which plays a crucial role in the conversion of glycogen to glucose-1-phosphate, is a target for therapeutic intervention in diabetes. In this study, we report the design and synthesis of 29 new derivatives of 2-oxo-1,2-dihydro pyridin-3-yl amides, as potential inhibitors of GP. The hit rate (45%) was high with 13 compounds inhibiting GPa (between 33% at 4.40 mM and an IC50 of 1.92 μM). Two lead compounds were identified as compounds exhibiting good GPa inhibition (IC50 = 2.1 and 1.92 μM). SAR analysis of these compounds revealed sensitivity of GPa to the length of the 2-oxo-1,2-dihydro pyridin-3-yl amide derivative and a preference for inclusion of a 3,4-dichlorobenzyl moiety.

ChemInform Abstract: Synthesis of N4‐Aryl‐β‐D‐glucopyranosylcytosines: A Methodology Study.

Abstract A number of leaving groups, including arylsulfonates, triazoles, 3-nitrotriazoles, and tetrazoles, have been studied for the substitution reaction by aryl and alkyl amines at the 4-position of β- d -glucopyranosyluracils. Examination of the stability, ease of purification and reactivity in the substitution reaction led to a number of optimized conditions with the most convenient involving substitution of triazole derivatives under microwave conditions in the presence of silica gel. Under these conditions, a number of N4-aryl-substituted β- d -glucopyranosylcytosines were prepared as potential inhibitors of glycogen phosphorylase, a molecular target for type-2 diabetes mellitus.

C-Glucopyranosyl-1,2,4-triazol-5-ones: synthesis and inhibition of glycogen phosphorylase

Various C-glucopyranosyl-1,2,4-triazolones were designed as potential inhibitors of glycogen phosphorylase. Syntheses of these compounds were performed with O-perbenzoylated glucose derivatives as precursors. High temperature ring closure of N1-carbamoyl-C-β-D-glucopyranosyl formamidrazone gave 3-β-D-glucopyranosyl-1,2,4-triazol-5-one. Reaction of N1-tosyl-C-β-D-glucopyranosyl formamidrazone with ClCOOEt furnished 3-β-D-glucopyranosyl-1-tosyl-1,2,4-triazol-5-one. In situ prepared β-D-glucopyranosylcarbonyl isocyanate was transformed by PhNHNHBoc into 3-β-D-glucopyranosyl-1-phenyl-1,2,4-triazol-5-one, while the analogous 1-(2-naphthyl) derivative was obtained from the unsubstituted triazolone by naphthalene-2-boronic acid in a Cu(II) catalyzed N-arylation. Test compounds were prepared by Zemplén deacylation. The new glucose derivatives had weak or no inhibition of rabbit muscle glycogen phosphorylase b: the best inhibitor was 3-β-D-glucopyranosyl-1-(2-naphthyl)-1,2,4-triazol-5-one (Ki = 80 µM).

Synthesis of N4-aryl-β-d-glucopyranosylcytosines: a methodology study

A number of leaving groups, including arylsulfonates, triazoles, 3-nitrotriazoles, and tetrazoles, have been studied for the substitution reaction by aryl and alkyl amines at the 4-position of β-d-glucopyranosyluracils. Examination of the stability, ease of purification and reactivity in the substitution reaction led to a number of optimized conditions with the most convenient involving substitution of triazole derivatives under microwave conditions in the presence of silica gel. Under these conditions, a number of N4-aryl-substituted β-d-glucopyranosylcytosines were prepared as potential inhibitors of glycogen phosphorylase, a molecular target for type-2 diabetes mellitus.

Synthesis, screening and docking of small heterocycles as Glycogen Phosphorylase inhibitors

A series of morpholine substituted amino acids (phenylalanine, leucine, lysine and glutamic acid) was synthesized. A fragment-based screening approach was then used to evaluate a series of small heterocycles, including morpholine, oxazoline, dihydro-1,3-oxazine, tetrahydro-1,3-oxazepine, thiazoline, tetrahydro-1,3-pyrimidine, tetrahydro-1,3-diazepine and hexahydro-1H-benzimidazole, as potential inhibitors of Glycogen Phosphorylase a. Thiazoline 7 displayed an improved potency (IC50 of 25 μM) and had good LE and LELP values, as compared to heterocycles 1, 5, 9–13 and 19 (IC50 values of 1.1 mM–23.9 mM). A docking study using the crystal structure of human liver Glycogen Phosphorylase, provided insight into the interactions of heterocycles 5, 7, 9–13 and 19 with Glycogen Phosphorylase.

Synthesis of C-xylopyranosyl- and xylopyranosylidene-spiro-heterocycles as potential inhibitors of glycogen phosphorylase

New derivatives of d-xylose with aglycons of the most efficient glucose derived inhibitors of glycogen phosphorylase were synthesized to explore the specificity of the enzyme towards the structure of the sugar part of the molecules. Thus, 2-(β-d-xylopyranosyl)benzimidazole and 3-substituted-5-(β-d-xylopyranosyl)-1,2,4-triazoles were obtained in multistep procedures from O-perbenzoylated β-d-xylopyranosyl cyanide. Cycloadditions of nitrile-oxides and O-peracetylated exo-xylal obtained from the corresponding β-d-xylopyranosyl cyanide furnished xylopyranosylidene-spiro-isoxazoline derivatives. Oxidative ring closure of O-peracetylated β-d-xylopyranosyl-thiohydroximates prepared from 1-thio-β-d-xylopyranose and nitrile-oxides gave xylopyranosylidene-spiro-oxathiazoles. The fully deprotected test compounds were assayed against rabbit muscle glycogen phosphorylase b to show moderate inhibition for 3-(2-naphthyl)-5-(β-d-xylopyranosyl)-1,2,4-triazole (IC50=0.9mM) only.

C-(β-d-Glucopyranosyl)formamidrazones, formic acid hydrazides and their transformations into 3-(β-d-glucopyranosyl)-5-substituted-1,2,4-triazoles: a synthetic and computational study

Synthesis of O-perbenzoylated 3-(β-d-glucopyranosyl)-5-substituted-1,2,4-triazoles, precursors of potent inhibitors of glycogen phosphorylase, was studied by ring closures of N1-acyl-carboxamidrazone type intermediates. Reactions of C-(β-d-glucopyranosyl)formimidate or C-(β-d-glucopyranosyl)formamidine with acid hydrazides as well as acylation of C-(β-d-glucopyranosyl)formamidrazone by acid chlorides unexpectedly gave the corresponding 1,3,4-oxadiazoles instead of 1,2,4-triazoles. The desired triazoles were obtained in reactions of C-(β-d-glucopyranosyl)formamidine or C-(β-d-glucopyranosyl)formyl chloride with arenecarboxamidrazones, and also in acylations of N1-tosyl-C-(β-d-glucopyranosyl)formamidrazone with acid chlorides. Theoretical calculations (B3LYP and M06-2X DFT with the standard 6-31G(d,p) basis set) on simple model compounds with methyl and phenyl substituents to understand the bifurcation of the ring closure of N1-acyl-carboxamidrazones indicated that in general the reaction led to 1,2,4-triazoles. However, the probability of the 1,3,4-oxadiazole forming pathway was shown to be significantly higher with N1-benzoyl-acetamidrazones, which were closest analogues of the intermediates resulting in C-glucosyl-1,3,4-oxadiazoles. It was thereby demonstrated that the substitution pattern of the N1-acyl-carboxamidrazones played a fundamental role in determining the direction of the ring closing reaction.

Synthesis of 5-halogenated 1,2,3-triazoles under stoichiometric Cu(I)-mediated azide–alkyne cycloaddition (CuAAC or ‘Click Chemistry’)

Glucosylated heterocycles have been identified as potent inhibitors of glycogen phosphorylase (GP), a biomolecular target for the treatment of hyperglycemia and therefore type 2 diabetes. Several glucosylated triazoles have been evaluated as GP inhibitors and additional structures are being considered in the present study with the introduction of a substituent at the 5-position of the triazole ring. The 1,3-dipolar cycloaddition of azide and alkyne using stoichiometric amounts of Cu(I) halides favored the formation of the 5-halogenated 1,2,3-triazoles. The influence of the copper halide introduced (CuI, CuBr, or CuCl) provided different results and more specifically for the CuCl system which afforded a dimeric 5,5′-bistriazole in good yield (56%) as evidenced by crystallographic data.