Glucopyranosylidene-spiro-imidazolinones, a New Ring System: Synthesis and Evaluation as Glycogen Phosphorylase Inhibitors by Enzyme Kinetics and X-ray Crystallography.

Epimeric series of aryl-substituted glucopyranosylidene-spiro-imidazolinones, an unprecedented new ring system, were synthesized from the corresponding Schiff bases of O-perbenzoylated (gluculopyranosylamine)onamides by intramolecular ring closure of the aldimine moieties with the carboxamide group elicited by N-bromosuccinimide in pyridine. Test compounds were obtained by Zemplén O-debenzoylation. Stereochemistry and ring tautomers of the new compounds were investigated by NMR, time-dependent density functional theory (TDDFT)-electronic circular dichroism, and DFT-NMR methods. Kinetic studies with rabbit muscle and human liver glycogen phosphorylases showed that the (R)-imidazolinones were 14-216 times more potent than the (S) epimers. The 2-naphthyl-substituted (R)-imidazolinone was the best inhibitor of the human enzyme (Ki 1.7 µM) and also acted on HepG2 cells (IC50 177 µM). X-ray crystallography revealed that only the (R) epimers bound in the crystal. Their inhibitory efficacy is based on the hydrogen-bonding interactions of the carbonyl oxygen and the NH of the imidazolinone ring.

High Consistency of Structure-Based Design and X-Ray Crystallography: Design, Synthesis, Kinetic Evaluation and Crystallographic Binding Mode Determination of Biphenyl-N-acyl-ß-d-Glucopyranosylamines as Glycogen Phosphorylase Inhibitors.

Structure-based design and synthesis of two biphenyl-N-acyl-ß-d-glucopyranosylamine derivatives as well as their assessment as inhibitors of human liver glycogen phosphorylase (hlGPa, a pharmaceutical target for type 2 diabetes) is presented. X-ray crystallography revealed the importance of structural water molecules and that the inhibitory efficacy correlates with the degree of disturbance caused by the inhibitor binding to a loop crucial for the catalytic mechanism. The in silico-derived models of the binding mode generated during the design process corresponded very well with the crystallographic data.

A multidisciplinary study of 3-(ß-d-glucopyranosyl)-5-substituted-1,2,4-triazole derivatives as glycogen phosphorylase inhibitors: Computation, synthesis, crystallography and kinetics reveal new potent inhibitors.

3-(ß-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 Ki'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-ß-d-glucopyranosyl tetrazole by N-benzyl-arenecarboximidoyl chlorides, ring closure of C-(ß-d-glucopyranosyl)formamidrazone with aroyl chlorides, or that of N-(ß-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.

Probing the ß-pocket of the active site of human liver glycogen phosphorylase with 3-(C-ß-d-glucopyranosyl)-5-(4-substituted-phenyl)-1, 2, 4-triazole inhibitors.

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 ß-1 substituent long enough to exploit interactions with a cavity adjacent to the active site, termed the ß-pocket. Recently, C-ß-d-glucopyranosyl imidazoles and 1, 2, 4-triazoles proved to be the best known glucose derived inhibitors of hlGP. Here we probe the ß-pocket by studying the inhibitory effect of six different groups at the para position of 3-(ß-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 Ki value of 0.43 µM. Structural studies have revealed the physicochemical diversity of the ß-pocket providing information for future rational inhibitor design studies.

Affinity Crystallography Reveals the Bioactive Compounds of Industrial Juicing Byproducts of Punica granatum for Glycogen Phosphorylase.

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.

Nanomolar Inhibitors of Glycogen Phosphorylase Based on ß-d-Glucosaminyl Heterocycles: A Combined Synthetic, Enzyme Kinetic, and Protein Crystallography Study.

Aryl substituted 1-(ß-d-glucosaminyl)-1,2,3-triazoles as well as C-ß-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-ß-d-glucosaminyl 1,2,3-triazoles showed weak or no inhibition, the C-ß-d-glucosaminyl derivatives had potent activity, and the best inhibitor was the 2-(ß-d-glucosaminyl)-4(5)-(2-naphthyl)-imidazole with a Ki 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.

Oxidation of human serum albumin exhibits inter-individual variability after an ultra-marathon mountain race.

The aim of this study was to examine the oxidation of human serum albumin (HSA) caused by oxidative stress following exhaustive and demanding exercise, such as an ultra-marathon race. For this purpose, blood samples from 12 adult runners who underwent a 103 km mountain ultra-marathon race were collected before the race, and also at 24, 48 and 72 h post-race. HSA was partially purified using affinity chromatography and consequently subjected to western blot analysis in order to determine the levels of disulfide dimers indicating oxidation. For reasons of comparison, the results were correlated with those from a previous study, in which the same samples were analyzed using different oxidative stress markers. The results revealed a good correlation between albumin dimers and protein carbonyls at all time points, while there was also a significant correlation with static oxidation reduction potential at 24 h, and a negative correlation with capacity oxidation reduction potential at 24 and 48 h. In addition, an individual analysis of albumin dimers exhibited great inter-individual differences, indicating the variation of HSA oxidation between different athletes. Namely, in some athletes, HSA seemed to be the main oxidation target of serum proteins, while in other athletes, there was even a reduction of HSA. This inter-individual variability in the oxidation of HSA may suggest that different interventions (e.g., through diet) may be required in order to confront the effects on athletes following strenuous exercise. On the whole, this study suggests the importance of the assessment of albumin dimers as a predictive marker for exercise-induced oxidative stress.

van der Waals interactions govern C-ß-d-glucopyranosyl triazoles' nM inhibitory potency in human liver glycogen phosphorylase.

3-(C-Glucopyranosyl)-5aryl-1,2,4-triazoles with an aryl moiety larger than phenyl have been shown to have strong inhibitory potency (Ki 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.

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.

The ammonium sulfate inhibition of human angiogenin.

In this study, we investigate the inhibition of human angiogenin by ammonium sulfate. The inhibitory potency of ammonium sulfate for human angiogenin (IC50 = 123.5 ± 14.9 mm) is comparable to that previously reported for RNase A (119.0 ± 6.5 mm) and RNase 2 (95.7 ± 9.3 mm). However, analysis of two X-ray crystal structures of human angiogenin in complex with sulfate anions (in acidic and basic pH environments, respectively) indicates an entirely distinct mechanism of inhibition. While ammonium sulfate inhibits the ribonucleolytic activity of RNase A and RNase 2 by binding to the active site of these enzymes, sulfate anions bind only to peripheral substrate anion-binding subsites of human angiogenin, and not to the active site.

Synthetic, enzyme kinetic, and protein crystallographic studies of C-ß-d-glucopyranosyl pyrroles and imidazoles reveal and explain low nanomolar inhibition of human liver glycogen phosphorylase.

C-ß-d-Glucopyranosyl pyrrole derivatives were prepared in the reactions of pyrrole, 2-, and 3-aryl-pyrroles with O-peracetylated ß-d-glucopyranosyl trichloroacetimidate, while 2-(ß-d-glucopyranosyl) indole was obtained by a cross coupling of O-perbenzylated ß-d-glucopyranosyl acetylene with N-tosyl-2-iodoaniline followed by spontaneous ring closure. An improved synthesis of O-perbenzoylated 2-(ß-d-glucopyranosyl) imidazoles was achieved by reacting C-glucopyranosyl formimidates with α-aminoketones. The deprotected compounds were assayed with isoforms of glycogen phosphorylase (GP) to show no activity of the pyrroles against rabbit muscle GPb. The imidazoles proved to be the best known glucose derived inhibitors of not only the muscle enzymes (both a and b) but also of the pharmacologically relevant human liver GPa (Ki = 156 and 26 nM for the 4(5)-phenyl and -(2-naphthyl) derivatives, respectively). An X-ray crystallographic study of the rmGPb-imidazole complexes revealed structural features of the strong binding, and also allowed to explain the absence of inhibition for the pyrrole derivatives.

Glycogen phosphorylase as a target for type 2 diabetes: synthetic, biochemical, structural and computational evaluation of novel N-acyl-N´-(ß-D-glucopyranosyl) urea inhibitors.

Glycogen phosphorylase (GP), a validated target for the development of anti-hyperglycaemic agents, has been targeted for the design of novel glycopyranosylamine inhibitors. Exploiting the two most potent inhibitors from our previous study of N-acyl-ß-D-glucopyranosylamines (Parmenopoulou et al., Bioorg. Med. Chem. 2014, 22, 4810), we have extended the linking group to -NHCONHCO- between the glucose moiety and the aliphatic/aromatic substituent in the GP catalytic site ß-cavity. The N-acyl-N´-(ß-D-glucopyranosyl) urea inhibitors were synthesized and their efficiency assessed by biochemical methods, revealing inhibition constant values of 4.95 µM and 2.53 µM. Crystal structures of GP in complex with these inhibitors were determined and analyzed, providing data for further structure based design efforts. A novel Linear Response - Molecular Mechanics Coulomb Surface Area (LR-MM-CBSA) method has been developed which relates predicted and experimental binding free energies for a training set of N-acyl-N´-(ß-D-glucopyranosyl) urea ligands with a correlation coefficient R(2) of 0.89 and leave-one-out cross-validation (LOO-cv) Q(2) statistic of 0.79. The method has significant applications to direct future lead optimization studies, where ligand entropy loss on binding is revealed as a key factor to be considered. ADMET property predictions revealed that apart from potential permeability issues, the synthesized N-acyl-N´-(ß-D-glucopyranosyl) urea inhibitors have drug-like potential without any toxicity warnings.

Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b.

We present a study on the binding of gallic acid and its dimer ellagic acid to glycogen phosphorylase (GP). Ellagic acid is a potent inhibitor with Kis of 13.4 and 7.5 µM, in contrast to gallic acid which displays Kis of 1.7 and 3.9 mM for GPb and GPa, respectively. Both compounds are competitive inhibitors with respect to the substrate, glucose-1-phoshate, and non-competitive to the allosteric activator, AMP. However, only ellagic acid functions with glucose in a strongly synergistic mode. The crystal structures of the GPb-gallic acid and GPb-ellagic acid complexes were determined at high resolution, revealing that both ligands bind to the inhibitor binding site of the enzyme and highlight the structural basis for the significant difference in their inhibitory potency.

Biochemical and biological assessment of the inhibitory potency of extracts from vinification byproducts of Vitis vinifera extracts against glycogen phosphorylase.

The inhibitory potency of thirteen polyphenolic extracts obtained from vinification byproducts of Greek varieties of Vitis vinifera against glycogen phosphorylase (GP) has been studied by kinetic experiments. GP is an enzyme involved in glucose homeostasis and a molecular target for the discovery of new hypoglycemic agents. Studies have shown that all extracts display significant inhibitory potency for GP in vitro with IC50 values in the range of low µg/mL. X-ray crystallographic analysis of GP crystals soaked with two of these extracts revealed that the most active ingredient is quercetin which binds at novel binding site, distinct from the other known sites of the enzyme. One of the most potent of the studied extracts had also a moderate effect on glycogenolysis in the cellular lever with an IC50 value of 17.35 µg/mL. These results highlight the importance of natural resources in the quest for the discovery of new hypoglycemic agents, while at the same time they can serve as the starting point for their exploitation for antidiabetic usage and the development of novel biofunctional foods.