Glycogen phosphorylase revisited: extending the resolution of the R- and T-state structures of the free enzyme and in complex with allosteric activators.

The crystal structures of free T-state and R-state glycogen phosphorylase (GP) and of R-state GP in complex with the allosteric activators IMP and AMP are reported at improved resolution. GP is a validated pharmaceutical target for the development of antihyperglycaemic agents, and the reported structures may have a significant impact on structure-based drug-design efforts. Comparisons with previously reported structures at lower resolution reveal the detailed conformation of important structural features in the allosteric transition of GP from the T-state to the R-state. The conformation of the N-terminal segment (residues 7-17), the position of which was not located in previous T-state structures, was revealed to form an α-helix (now termed α0). The conformation of this segment (which contains Ser14, phosphorylation of which leads to the activation of GP) is significantly different between the T-state and the R-state, pointing in opposite directions. In the T-state it is packed between helices α4 and α16 (residues 104-115 and 497-508, respectively), while in the R-state it is packed against helix α1 (residues 22'-38') and towards the loop connecting helices α4' and α5' of the neighbouring subunit. The allosteric binding site where AMP and IMP bind is formed by the ordering of a loop (residues 313-326) which is disordered in the free structure, and adopts a conformation dictated mainly by the type of nucleotide that binds at this site.

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.

Affinity Crystallography Reveals Binding of Pomegranate Juice Anthocyanins at the Inhibitor Site of Glycogen Phosphorylase: The Contribution of a Sugar Moiety to Potency and Its Implications to the Binding Mode.

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.

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.

Glycogen phosphorylase of shrimp (Litopenaeus vannamei): Structure, expression and anti-WSSV function.

Glycogen phosphorylase (GP, EC 2.4.1.1) catalyze the rate-limiting step in glycogenolysis in animals, forming glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond. Therefore, GP plays a crucial role in carbohydrate metabolism. In the present study, the full-length cDNA sequence of GP (LvGP) was cloned from shrimp, Litopenaeus vannamei. The obtained 3242-bp LvGP cDNA sequence included a 5'-terminal untranslated region (UTR) of 48 bp, an open reading frame (ORF) of 2559 bp encoding a polypeptide of 852 amino acids (aa) and a 3'-UTR of 635 bp. The predicted LvGP protein sequence contained a typical phosphorylase domain (113-829 aa) and shared 72%-97% identities with GP from other species. Phylogenetic analysis revealed that LvGP showed the closest relationship with GP from Marsupenaeus japonicus. Tissue expression profiles showed that LvGP existed in most examined tissues, with the most predominant expression in the brain, followed by the muscles and stomach. LvGP transcripts in hepatopancreas and hemocytes were up regulated after the WSSV challenge. Furthermore, the role of LvGP in shrimp defending against WSSV infection was investigated by RNA interference (RNAi). Our findings showed that WSSV proliferation and shrimp accumulative mortality increased significantly after LvGP RNAi (P < 0.01). The glycogen, glucose, and pyruvate content decreased in GP RNAi shrimp after WSSV injection, however, the lactate and ATP concentration enhanced. Moreover, lectin and anti-lipopolysaccharide factor2 (ALF2) were induced in LvGP silencing shrimp after WSSV infection, whereas the expression levels of crustin, ALF1 and ALF3 decreased. Our results suggested that the LvGP might play a crucial role in shrimp defending against WSSV infection by regulating metabolism and affecting the anti-infectious gene expression.

Chemoproteomic fishing identifies arzanol as a positive modulator of brain glycogen phosphorylase.

The interactome of arzanol was investigated by MS-based chemical proteomics, a pioneering technology for small molecule target discovery. Brain glycogen phosphorylase (bGP), a key regulator of glucose metabolism so far refractory to small molecule modulation, was identified as the main high-affinity target of arzanol. Competitive affinity-based proteomics, DARTS, molecular docking, surface plasmon resonance and in vitro biological assays provided molecular mechanistic insights into the arzanol-enzyme interaction, qualifying this positive modulator of bGP for further studies in the realm of neurodegeneration and cancer.

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.

Probing the catalytic site of rabbit muscle glycogen phosphorylase using a series of specifically modified maltohexaose derivatives.

Glycogen phosphorylase (GP) is an allosteric enzyme whose catalytic site comprises six subsites (SG1, SG-1, SG-2, SG-3, SG-4, and SP) that are complementary to tandem five glucose residues and one inorganic phosphate molecule, respectively. In the catalysis of GP, the nonreducing-end glucose (Glc) of the maltooligosaccharide substrate binds to SG1 and is then phosphorolyzed to yield glucose 1-phosphate. In this study, we probed the catalytic site of rabbit muscle GP using pyridylaminated-maltohexaose (Glcα1-4Glcα1-4Glcα1-4Glcα1-4Glcα1-4GlcPA, where GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol]; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (Glc m -AltNAc-Glc n -GlcPA, where m + n = 4 and AltNAc is 3-acetoamido-3-deoxy-D-altrose). PA-0 served as an efficient substrate for GP, whereas the other PA-0 derivatives were not as good as the PA-0, indicating that substrate recognition by all the SG1 -SG-4 subsites was important for the catalysis of GP. By comparing the initial reaction rate toward the PA-0 derivatives (V derivative) with that toward PA-0 (V PA-0), we found that the value of V derivative/V PA-0 decreased significantly as the level of allosteric activation of GP increased. These results suggest that some conformational changes have taken place in the maltooligosaccharide-binding region of the GP catalytic site during allosteric regulation.

Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide-Protein Interactions.

A 42-residue polypeptide conjugated to a small-molecule organic ligand capable of targeting the phosphorylated side chain of Ser15 was shown to bind glycogen phosphorylase a (GPa) with a KD value of 280 nm. The replacement of hydrophobic amino acids by Ala reduced affinities, whereas the incorporation of l-2-aminooctanoic acid (Aoc) increased them. Replacing Nle5, Ile9 and Leu12 by Aoc reduced the KD value from 280 to 27 nm. "Downsizing" the 42-mer to an undecamer gave rise to an affinity for GPa an order of magnitude lower, but the undecamer in which Nle5, Ile9 and Leu12 were replaced by Aoc showed a KD value of 550 nm, comparable with that of the parent 42-mer. The use of Aoc residues offers a convenient route to increased affinity in protein recognition as well as a strategy for the "downsizing" of peptides essentially without loss of affinity. The results show that hydrophobic binding sites can be found on protein surfaces by comparing the affinities of polypeptide conjugates in which Aoc residues replace Nle, Ile, Leu or Phe with those of their unmodified counterparts. Polypeptide conjugates thus provide valuable opportunities for the optimization of peptides and small organic compounds in biotechnology and biomedicine.

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 structure of brain glycogen phosphorylase-from allosteric regulation mechanisms to clinical perspectives.

Glycogen phosphorylase (GP) is the key enzyme that regulates glycogen mobilization in cells. GP is a complex allosteric enzyme that comprises a family of three isozymes: muscle GP (mGP), liver GP (lGP), and brain GP (bGP). Although the three isozymes display high similarity and catalyze the same reaction, they differ in their sensitivity to the allosteric activator adenosine monophosphate (AMP). Moreover, inactivating mutations in mGP and lGP have been known to be associated with glycogen storage diseases (McArdle and Hers disease, respectively). The determination, decades ago, of the structure of mGP and lGP have allowed to better understand the allosteric regulation of these two isoforms and the development of specific inhibitors. Despite its important role in brain glycogen metabolism, the structure of the brain GP had remained elusive. Here, we provide an overview of the human brain GP structure and its relationship with the two other members of this key family of the metabolic enzymes. We also summarize how this structure provides valuable information to understand the regulation of bGP and to design specific ligands of potential pharmacological interest.

Sourcing the affinity of flavonoids for the glycogen phosphorylase inhibitor site via crystallography, kinetics and QM/MM-PBSA binding studies: comparison of chrysin and flavopiridol.

Flavonoids have been discovered as novel inhibitors of glycogen phosphorylase (GP), a target to control hyperglycemia in type 2 diabetes. To elucidate the mechanism of inhibition, we have determined the crystal structure of the GPb-chrysin complex at 1.9 Å resolution. Chrysin is accommodated at the inhibitor site intercalating between the aromatic side chains of Phe285 and Tyr613 through π-stacking interactions. Chrysin binds to GPb approximately 15 times weaker (Ki=19.01 µM) than flavopiridol (Ki=1.24 µM), exclusively at the inhibitor site, and both inhibitors display similar behavior with respect to AMP. To identify the source of flavopiridols' stronger affinity, molecular docking with Glide and postdocking binding free energy calculations using QM/MM-PBSA have been performed and compared. Whereas docking failed to correctly rank inhibitor binding conformations, the QM/MM-PBSA method employing M06-2X/6-31+G to model the π-stacking interactions correctly reproduced the experimental results. Flavopiridols' greater binding affinity is sourced to favorable interactions of the cationic 4-hydroxypiperidin-1-yl substituent with GPb, with desolvation effects limited by the substituent conformation adopted in the crystallographic complex. Further successful predictions using QM/MM-PBSA for the flavonoid quercetagetin (which binds at the allosteric site) leads us to propose the methodology as a useful and inexpensive tool to predict flavonoid binding.

The binding of C5-alkynyl and alkylfurano[2,3-d]pyrimidine glucopyranonucleosides to glycogen phosphorylase b: synthesis, biochemical and biological assessment.

C5-alkynyl and alkylfurano[2,3-d]pyrimidine glucopyranonucleosides have been synthesized and studied as inhibitors of glycogen phosphorylase b (GPb). Kinetic experiments have shown that most of these compounds were low micromolar inhibitors of the enzyme. The best inhibitor was 1-(ß-D-glucopyranosyl)-5-ethynyluracil (K(i)=4.7 µM). Crystallographic analysis of these compounds in complex with GPb revealed that inhibitors with a long C5-alkynyl group exploited interactions with ß-pocket of the active site and induced significant conformational changes of the 280s loop compared to GPb in complex with compounds with a short C5-alkynyl group. The results highlight the importance in the length of the aliphatic groups used to enhance inhibitory potency for the exploitation of the hydrophobic ß-pocket. The best of the inhibitors had also a moderate effect on glycogenolysis in the cellular lever with an IC(50) value of 291.4 µM.

Ligand-based modelling followed by synthetic exploration unveil novel glycogen phosphorylase inhibitory leads.

Glycogen phosphorylase (GP) is a valid anti-diabetic target. Accordingly, we applied a drug discovery workflow to unveil novel inhibitory GP leads via combining pharmacophore modeling, QSAR analysis and in silico screening, followed by synthetic exploration of active hits. Virtual screening identified six low micromolar inhibitory leads from the National Cancer Institute (NCI) list of compounds. The most potent hits exhibited anti-GP IC(50) values of 3.2 and 4.1 µM. Synthetic exploration of hit 59 (IC(50)=4.1 µM) yielded 25 lead inhibitors with the best illustrating IC(50) of 3.0 µM. Interestingly, we prepared several novel mixed oxalyl amide anti-GP leads employing new chemical reaction involving succinic acid-based adducts.

Small peptides released from muscle glycolytic enzymes during dry-cured ham processing.

Glycolytic enzymes are a group of sarcoplasmic enzymes responsible for the extraction of the energy available from carbohydrates. The glycolytic pathway consists of 10 enzyme-catalyzed steps. Fragments identified in this study, within the range 1100-2600 Da, correspond to glycogen phosphorylase enzyme, which catalyzes the rate limiting step in the degradation of glycogen, enzymes that catalyze steps 6-10 of glycolysis (glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase, respectively), and lactate dehydrogenase, which catalyzes the interconversion of pyruvate and lactate. A total of 45 specific fragments of these enzymes resulting from the processing of dry-cured ham are reported for the first time in this work. This study evidences the intense proteolysis occurring in the sarcoplasmic fraction of dry-cured ham as well as facilitates the choice of the most adequate tools in the identification of naturally generated peptides through comparison between Paragon and Mascot search engines, together with UniProt and NCBInr databases.

Naturally occurring pentacyclic triterpenes as inhibitors of glycogen phosphorylase: synthesis, structure-activity relationships, and X-ray crystallographic studies.

Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this study, were biologically evaluated as inhibitors of rabbit muscle glycogen phosphorylase a (GPa). From SAR studies, the presence of a sugar moiety in triterpene saponins resulted in a markedly decreased activity ( 7, 18- 20) or no activity ( 21, 22). These saponins, however, might find their value as potential natural prodrugs which are much more water-soluble than their corresponding aglycones. To elucidate the mechanism of GP inhibition, we have determined the crystal structures of the GPb-asiatic acid and GPb-maslinic acid complexes. The X-ray analysis indicates that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Pentacyclic triterpenes represent a promising class of multiple-target antidiabetic agents that exert hypoglycemic effects, at least in part, through GP inhibition.

Modeling and informatics in designing anti-diabetic agents.

Diabetes mellitus is a chronic metabolic disorder, characterized by glucose overproduction and glucose underutilization. Current therapy for T2DM includes drugs, like metformin, glitazones, sulphonyl ureas, etc. Extensive research has been carried out world wide on molecular targets for T2DM like PPARgamma, PTP1B, DPP-IV, GSK-3, cannabinoid receptor, fructose-bisphosphatases, beta3 adrenoceptor, etc. in the development of newer anti-diabetic agents. These therapeutic targets are quite important and most of them are suitable for in silico analysis. Hence, many molecular modeling and informatics studies like, molecular docking, pharmacophore mapping, 3D-QSAR, virtual screening, quantum chemical studies, and pharmacoinformatics like bioinformatics and chemoinformatics studies have been performed on the drugs/leads/targets associated with T2DM. Several of these in silico efforts are exemplary studies; the methodologies adopted in these studies can be emulated in many other therapeutic areas. A review of the rational approaches reported in designing anti-diabetic agents is presented in this article.

Modeling aided design of potent glycogen phosphorylase inhibitors.

Molecular modeling has been used to assist in the development of a novel series of potent glycogen phosphorylase inhibitors based on a phenyl diacid lead, compound 1. In the absence of suitable competitive binding assays, compound 1 was predicted to bind at the AMP allosteric site based on superposition onto known inhibitors which bind at different sites in the enzyme and analyses of the surrounding protein environment associated with these distinct sites. Possible docking modes of compound 1 at the AMP allosteric site were further explored using the crystal structure of rabbit muscle glycogen phosphorylase complexed with a Bayer diacid compound W1807 (PDB entry 3AMV). Compound 1 was predicted to interact with positively charged arginines at the AMP allosteric site in the docking model. Characterization of the binding pocket by a grid-based surface calculation of the docking model revealed a large unfilled hydrophobic region near the central phenyl ring, suggesting that compounds with larger hydrophobic groups in this region would improve binding. A series of naphthyl diacid compounds were designed and synthesized to access this hydrophobic cleft, and showed significantly improved potency.

Crystal structure of glycogen synthase: homologous enzymes catalyze glycogen synthesis and degradation.

Glycogen and starch are the major readily accessible energy storage compounds in nearly all living organisms. Glycogen is a very large branched glucose homopolymer containing about 90% alpha-1,4-glucosidic linkages and 10% alpha-1,6 linkages. Its synthesis and degradation constitute central pathways in the metabolism of living cells regulating a global carbon/energy buffer compartment. Glycogen biosynthesis involves the action of several enzymes among which glycogen synthase catalyzes the synthesis of the alpha-1,4-glucose backbone. We now report the first crystal structure of glycogen synthase in the presence and absence of adenosine diphosphate. The overall fold and the active site architecture of the protein are remarkably similar to those of glycogen phosphorylase, indicating a common catalytic mechanism and comparable substrate-binding properties. In contrast to glycogen phosphorylase, glycogen synthase has a much wider catalytic cleft, which is predicted to undergo an important interdomain 'closure' movement during the catalytic cycle. The structures also provide useful hints to shed light on the allosteric regulation mechanisms of yeast/mammalian glycogen synthases.

Identification, synthesis, and characterization of new glycogen phosphorylase inhibitors binding to the allosteric AMP site.

Inhibition of glycogen phosphorylase (GP) has attracted considerable attention during the last five to 10 years as a means of treating the elevated hepatic glucose production seen in patients with type 2 diabetes. Several different GP inhibitors binding to various binding sites of the GP enzyme have been reported in the literature. In this paper we report on a novel class of compounds that have been identified as potent GP inhibitors. Their synthesis, mode of binding to the allosteric AMP site as well as in vitro data on GP inhibition are shown. The most potent inhibitor was found to be 4-[2,4-bis-(3-nitrobenzoylamino)phenoxy]phthalic acid (4j) with an IC(50) value of 74 nM. This compound together with a closely related analogue was further characterized by enzyme kinetics and in primary rat hepatocytes.