Glycogen phosphorylase inhibitors: a patent review (2008 - 2012).

INTRODUCTION: Glycogen phosphorylase (GP) is the enzyme responsible for the synthesis of glucose-1-phosphate, the source of energy for muscles and the rest of the body. The binding of different ligands in catalytic or allosteric sites assures activation and deactivation of the enzyme. A description of the regulation mechanism and the implications in glycogen metabolism are given. AREAS COVERED: Deregulation of GP has been observed in diseases such as diabetes mellitus or cancers. Therefore, it appears as an attractive therapeutic target for the treatment of such pathologies. Numbers of inhibitors have been published in academic literature or patented in the last two decades. This review presents the main patent claims published between 2008 and 2012. EXPERT OPINION: Good inhibitors with interesting IC50 and in vivo results are presented. However, such therapeutic strategy raises questions and some answers are proposed to bring new insights in the field.

[Optimization of enzymatic preparation of glucose 1-phosphate by response surface methodology].

With glucose as substrate, sodium tripolyphosphate as the phosphorus acylating agent, and phosphorylase of Solanum tuberosum as the catalyst, glucose 1-phosphate was synthesized. Based on a three-level, three-variable Box-Behnken experimental design, response surface methodology was used to evaluate the effects of temperature, molar ratio of glucose to sodium tripolyphosphate and time on the production. The structure of the product was confirmed by 1H NMR spectra. The results show that the optimum conditions were as follows: temperature 35 degrees C, molar ratio of glucose to sodium tripolyphosphate 1.35:1 and time 19 h.

Examining the role of phosphate in glycosyl transfer reactions of Cellulomonas uda cellobiose phosphorylase using D-glucal as donor substrate.

Cellobiose phosphorylase from Cellulomonas uda (CuCPase) is shown to utilize D-glucal as slow alternative donor substrate for stereospecific glycosyl transfer to inorganic phosphate, giving 2-deoxy-α-D-glucose 1-phosphate as the product. When performed in D(2)O, enzymatic phosphorolysis of D-glucal proceeds with incorporation of deuterium in equatorial position at C-2, implying a stereochemical course of reaction where substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism, which is supported by results of docking studies, involves direct protonation of D-glucal by the enzyme-bound phosphate, which then performs nucleophilic attack on the reactive C-1 of donor substrate. When offered D-glucose next to D-glucal and phosphate, CuCPase produces 2-deoxy-ß-D-glucosyl-(1→4)-D-glucose and 2-deoxy-α-D-glucose 1-phosphate in a ratio governed by mass action of the two acceptor substrates present. Enzymatic synthesis of 2-deoxy-ß-D-glucosyl-(1→4)-D-glucose is effectively promoted by catalytic concentrations of phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of CuCPase, D-glucal, phosphate, and D-glucose. Conversion of D-glucal and phosphate presents a convenient single-step synthesis of 2-deoxy-α-D-glucose 1-phosphate that is difficult to prepare chemically.

Characterization of the TDP-D-ravidosamine biosynthetic pathway: one-pot enzymatic synthesis of TDP-D-ravidosamine from thymidine-5-phosphate and glucose-1-phosphate.

Ravidomycin V and related compounds, e.g., FE35A-B, exhibit potent anticancer activities against various cancer cell lines in the presence of visible light. The amino sugar moieties (D-ravidosamine and its analogues, respectively) in these molecules contribute to the higher potencies of ravidomycin and analogues when compared to closely related compounds with neutral or branched sugars. Within the ravidomycin V biosynthetic gene cluster, five putative genes encoding NDP-D-ravidosamine biosynthetic enzymes were identified. Through the activities of the isolated enzymes in vitro, it is demonstrated that ravD, ravE, ravIM, ravAMT and ravNMT encode TDP-D-glucose synthase, TDP-4-keto-6-deoxy-D-glucose-4,6-dehydratase, TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase, TDP-3-keto-6-deoxy-D-galactose-3-aminotransferase, and TDP-3-amino-3,6-dideoxy-D-galactose-N,N-dimethyl-transferase, respectively. A protocol for a one-pot enzymatic synthesis of TDP-D-ravidosamine has been developed. The results presented here now set the stage to produce TDP-D-ravidosamine routinely for glycosylation studies.

Enzymatic production of ß-D-glucose-1-phosphate from trehalose.

ß-D-Glucose-1-phosphate (ßGlc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This article provides an efficient production method of ßGlc1P from trehalose and phosphate using the thermostable trehalose phosphorylase from Thermoanaerobacter brockii. At the process temperature of 60 °C, Escherichia coli expression host cells are lysed and cell treatment prior to the reaction is, therefore, not required. In this way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast, followed by chemical phosphate precipitation. In a next step, ßGlc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what is observed with other glycosylphosphates. After conversion of the product to its cyclohexylammonium salt, however, crystals could be readily obtained. Although both purification methods were quantitative (>99% recovery), a large amount of product (50%) was lost during crystallization. Nevertheless, a production process for crystalline ßGlc1P is now available from the cheap substrates trehalose and inorganic phosphate.

Recombinant sucrose phosphorylase from Leuconostoc mesenteroides: characterization, kinetic studies of transglucosylation, and application of immobilised enzyme for production of alpha-D-glucose 1-phosphate.

Sucrose phosphorylase catalyzes the reversible conversion of sucrose (alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside) and phosphate into D-fructose and alpha-D-glucose 1-phosphate. We report on the molecular cloning and expression of the structural gene encoding sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase) in Escherichia coli DH10B. The recombinant enzyme, containing an 11 amino acid-long N-terminal metal affinity fusion peptide, was overproduced 60-fold in comparison with the natural enzyme. It was purified to apparent homogeneity using copper-loaded Chelating Sepharose and obtained in 20% yield with a specific activity of 190 Umg(-1). LmSPase was covalently attached onto Eupergit C with a binding efficiency of 50% and used for the continuous production of alpha-D-glucose 1-phosphate from sucrose and phosphate (600 mM each) in a packed-bed immobilised enzyme reactor (30 degrees C, pH 7.0). The reactor was operated at a stable conversion of 91% (550 mM product) and productivity of approximately 11 gl(-1)h(-1) for up to 600 h. A kinetic study of transglucosylation by soluble LmSPase was performed using alpha-d-glucose 1-phosphate as the donor substrate and various alcohols as acceptors. D- and L-arabitol were found to be good glucosyl acceptors.

Bacillus subtilis alpha-phosphoglucomutase is required for normal cell morphology and biofilm formation.

Mutations designated gtaC and gtaE that affect alpha-phosphoglucomutase activity required for interconversion of glucose 6-phosphate and alpha-glucose 1-phosphate were mapped to the Bacillus subtilis pgcA (yhxB) gene. Backcrossing of the two mutations into the 168 reference strain was accompanied by impaired alpha-phosphoglucomutase activity in the soluble cell extract fraction, altered colony and cell morphology, and resistance to phages phi29 and rho11. Altered cell morphology, reversible by additional magnesium ions, may be correlated with a deficiency in the membrane glycolipid. The deficiency in biofilm formation in gtaC and gtaE mutants may be attributed to an inability to synthesize UDP-glucose, an important intermediate in a number of cell envelope biosynthetic processes.

Characterization of the essential gene glmM encoding phosphoglucosamine mutase in Escherichia coli.

Two different approaches to identify the gene encoding the phosphoglucosamine mutase in Escherichia coli were used: (i) the purification to near homogeneity of this enzyme from a wild type strain and the determination of its N-terminal amino acid sequence; (ii) the search in data bases of an E. coli protein of unknown function showing sequence similarities with other hexosephosphate mutase activities. Both investigations revealed the same open reading frame named yhbF located within the leuU-dacB region at 69.5 min on the chromosome (Dallas, W. S., Dev, I. K., and Ray, P. H. (1993) J. Bacteriol. 175, 7743-7744). The predicted 445-residue protein with a calculated mass of 47.5 kDa contained in particular a short region GIVISASHNP with high similarity to the putative active site of hexosephosphate mutases. In vitro assays showed that the overexpression of this gene in E. coli cells led to a significant overproduction (from 15- to 50-fold) of phosphoglucosamine mutase activity. A hexose 1,6-diphosphate-dependent phosphorylation of the enzyme, which probably involves the serine residue at position 102, is apparently required for its catalytic action. As expected, the inactivation of this gene, which is essential for bacterial growth, led to the progressive depletion of the pools of precursors located downstream from glucosamine 1-phosphate in the pathway for peptidoglycan synthesis. This was followed by various alterations of cell shape and finally cells were lysed when their peptidoglycan content decreased to a critical value corresponding to about 60% of its normal level. The gene for this enzyme, which is essential for peptidoglycan and lipopolysaccharide biosyntheses, has been designated glmM.

Gluconeogenesis and glucuronidation in liver in vivo and the heterogeneity of hepatocyte function.

In order to examine metabolic zonation in human liver, [2-14C]glycerol, which labels carbons 2 and 5 of glucose-6-P, and [1-14C]lactate, which labels carbons 3 and 4 of glucose-6-P, in the process of gluconeogenesis, were infused intravenously into healthy subjects who ingested acetaminophen and had fasted 36 h. Distributions of 14C were determined in glucose in blood and in the glucuronic acid moiety of acetaminophen glucuronide excreted in urine. Ratios of 14C in carbons 2 and 5 to 14C in carbons 3 and 4 were significantly higher in blood glucose than in glucuronide. Since glucose and glucuronic acid are formed from glucose-6-P in liver without randomization of carbon, the differences in the ratios indicate that the pool of glucose-6-P in liver is not homogeneous. The glucuronide sampled glucose-6-P with more label from lactate than glycerol compared to the glucose-6-P sampled by the glucose. The apparent explanation is the greater decrease in glycerol compared with lactate concentration as blood streams from the periportal to the perivenous zones of the liver lobule. Glucuronidation is then expressed in humans relatively more in the perivenous than periportal zones and gluconeogenesis from glycerol more in the periportal than perivenous zones.

The Cryptococcus neoformans GAL7 gene and its use as an inducible promoter.

A Cryptococcus neoformans galactose auxotroph was created by ultraviolet light mutagenesis and complemented with a C. neoformans genomic library. The translated sequence of the complementing DNA revealed a high degree of similarity to a number of UDP glucose-D-galactose-1-phosphate uridylyltransferases. Expression of C. neoformans GAL7 mRNA followed a pattern similar to Saccharomyces cerevisiae expression; it was first observed within 2.5 min of induction and fully induced by 30 min. The gene was completely repressed in the presence of glucose. The GAL7 promoter was isolated and used to construct a promoter cassette. Two genes were tested in this cassette for galactose regulation by creating GAL7 promoter fusions with their coding regions. MF alpha, which encodes a pheromone, was found to produce filaments only in transformants that were induced by galactose. A second gene, beta-glucuronidase (gusA), which is a commonly used reporter gene, was tested and also found to be expressed. When the GAL7p::GUS fusion was used to quantify inducibility of the GAL7 promoter, the level of enzyme activity was at least 500-fold greater for cells grown in galactose than for cells grown in glucose. The GAL7 promoter is the first inducible promoter characterized in C. neoformans and the GUS gene is the first heterologous gene shown to be expressed in this yeast pathogen.

Harry M. Vars Research Award. Influence of fasting on glutamine transport in rat liver.

During starvation, the liver switches from an organ of net glutamine uptake to an organ of net glutamine release to help maintain blood glutamine levels. We hypothesized that this shift in hepatic glutamine exchange was regulated at the level of the hepatocyte plasma membrane by adaptive changes in glutamine transport. To test this hypothesis, adult rats (200 g) were allowed to consume regular rat food ad libitum (fed, n = 8) or were fasted for 72 hours (fasted, n = 8, access to water allowed). Livers were excised and hepatocyte plasma membrane vesicles were prepared by differential and Percoll density gradient centrifugation. Vesicle purity and functionality were assessed by marker enzyme measurements, classic "overshoots," and time courses, which showed similar vesicle size. Uptake of 3H-glutamine by hepatocyte plasma membrane vesicles in the presence and absence of sodium was assayed by a rapid mixing/filtration method, which reflects actual transport across the hepatocyte cell membrane in vivo. Fasted rats lost 15 +/- 2% of body weight; fed rats gained weight. Na(+)-dependent glutamine transport (system "N," mediates uptake into the hepatocyte) fell by 22% in the starved group, indicating a diminished rate of glutamine transport into the hepatocyte. In contrast, carrier-mediated Na(+)-independent glutamine transport (system "n," mediates the release of glutamine out of the cell) doubled in the starved animals. Diffusion of glutamine across the vesicle membrane was unchanged by starvation.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic regulation of endoglucanase synthesis in Trichoderma reesei: participation of cyclic AMP and glucose-6-phosphate.

The synthesis of endoglucanase by young induced mycelia of Trichoderma reesei QM 9414 incubated in the presence of 1 mM sophorose (a potent cellulase inducer) was stimulated or repressed by additions of dibutyryl cyclic AMP (dBcAMP), depending on the concentration. At low concentrations (10(-6) and 10(-5) M), dBcAMP stimulated the formation of endoglucanase; higher concentrations of dBcAMP (10(-3) and 10(-2) M) repressed the synthesis of endoglucanase. Addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) at 1 and 10 microM concentrations to young induced mycelia caused an increase in intracellular cAMP and stimulated the production of endoglucanase. Neither exogenous dBcAMP nor IBMX was capable of inducing endoglucanase synthesis by itself, and neither was able to relieve catabolite repression of endoglucanase synthesis caused by glucose. All of the monosaccharides tested caused a more or less transient increase in intracellular cAMP. However, the effect of these treatments on endoglucanase synthesis was varied. The phosphorylable hexoses, both metabolizable and nonmetabolizable, increased the intracellular level of glucose-6-phosphate or its analogs and repressed endoglucanase synthesis. Nonphosphorylable sugars, such as 6-deoxyglucose, xylose, L-fucose, and (or) L-sorbose, did not influence the glucose-6-phosphate level and stimulated endoglucanase production to varying degrees. It is concluded that both cAMP and glucose-6-phosphate are involved in regulating cellulase synthesis in T. reesei. However, these factors seem to act in opposing directions.

Ethanol stimulates glycogenolysis in livers from fed rats.

To determine the reason for the lack of a hypoglycemic effect of ethanol in the fed state, the effect of ethanol on glucose turnover, liver glycogenolysis, and glucose metabolites was determined. Chronically catheterized awake and freely moving fed rats received either ethanol (blood ethanol, 37 +/- 10 mmol/liter, n = 11) or saline (n = 13) intravenously for 4 hr. Glucose turnover was determined using a primed continuous infusion of [3-3H]glucose. The liver was freeze clamped at 4 hr for glycogen and metabolite measurements. Plasma glucose (5.8 +/- 0.3 mmol/liter vs 6.3 +/- 0.2 mmol/liter at 4 hr, ethanol versus saline) and the rate of glucose turnover (61 +/- 9 vs 58 +/- 8 moles/kg.min) were similar during the ethanol and saline infusions. Plasma lactate was significantly higher in the ethanol (1.32 +/- 0.05 mmol/liter) than in the saline (0.86 +/- 0.06 mmol/liter, P less than 0.001) study. Concentrations of gluconeogenic intermediates in the liver (glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate, and pyruvate) were all significantly and -30% lower in ethanol-infused than in saline-infused rats. The liver citrate content was similar in ethanol-infused than in saline-infused rats. The liver citrate content was similar in ethanol (0.38 +/- 0.03 mmol/liter) and saline (0.37 +/- 0.04 mmol/liter) studies. Liver glycogen was 75% lower in the ethanol-infused (61 +/- 9 mmol/kg dry wt) than the saline (242 +/- 27 mmol/kg dry wt, P less than 0.001)-infused rats. These data demonstrate that in fed rats given ethanol, glucose turnover is maintained constant by accelerated glycogenolysis. Thus, inhibition of gluconeogenesis by ethanol does not lower hepatic glucose production unless compensatory glycogenolysis can be prevented.

Specificity of glucose 1,6-bisphosphate synthesis in rabbit skeletal muscle.

1. To compare glucose 1,6-bisphosphate synthesis in different types of cells, we partially purified (2000-fold) a glycerate 1,3 P2-dependent glucose 1,6-bisphosphate synthase from rabbit skeletal muscle. 2. In agreement with the results reported by others for mouse brain and pig skeletal muscle, the enzyme can be separated from bulk phosphoglucomutase (PGM) activity by DEAE-cellulose chromatography of crude cellular extract. This cannot be achieved on human hemolysates where glycerate 1,3-P2-dependent glucose 1,2-bisphosphate synthesis is displayed only by multifunctional PGM2 isoenzymes. 3. The Km values for glycerate 1,3-P2 (0.50 microM), glucose 1-phosphate (90 microM), Mg2+ (0.22 mM), and also pH optimum (7.8) and mol. wt (70,000) of the rabbit skeletal muscle enzyme are similar to those of the enzymes from mouse brain and human red blood cells, but they differ from those reported for the pig skeletal muscle enzyme.

[Synthesis of phosphodiesters of 2-acetamido-2-deoxy-D-glucose--fragments of polymers of capsules from Escherichia coli K51 and Neisseria meningitidis X]. / Sintez fosfodiéfirov 2-atsetamido-2-dezoksi-D-gliukozy--fragmentov polimerov kapsul Escherichia coli K51 i Neisseria meningitidis X.

Hydrogenphosphonate method was used for synthesis of 4-nitrophenyl 2-acetamido-3- and 4-nitrophenyl 2-acetamido-4-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate)-2-deoxy-beta-D-glucopyranosides. The glycosides, phosphate diester fragments of the title bacteria capsular antigens, were obtained by H-phosphorylation of the suitably protected 2-acetamido-2-deoxy-beta-D-glucopyranosides with 2-acetamido-3,4,6-tri-O-benzoyl-2-deoxy-alpha-D-glucopyranosyl H-phosphonate in the presence of trimethylacetyl chloride followed by oxidation and deprotection.

Antimicrobial properties of N3-(iodoacetyl)-L-2,3-diaminopropanoic acid-peptide conjugates.

Six peptide conjugates consisting of either norvaline, methionine, or lysine and N3-(iodoacetyl)-L-2,3-diaminopropanoic acid--a strong, irreversible inactivator of bacterial and fungal glucosamine-6-phosphate synthase--were synthesized and their antibacterial and antifungal activities were evaluated. Antimicrobial potencies of these peptides were correlated with their transport and cleavage rates inside the cells. Bacteriolysis of Bacillus pumilus cells and inhibition of [14C]glucose incorporation into cell-wall polysaccharides of Candida albicans as a result of glucosamine 6-phosphate inactivation were also observed. Reversal of growth inhibitory effect of these peptides by N-acetylglucosamine in bacteria and fungi suggests the effective delivery of N3-iodoacetyl-L-2,3-diaminopropanoic acid into the cell by a peptide-transport system.

Hexose metabolism in pancreatic islets: preferential utilization of mitochondrial ATP for glucose phosphorylation.

The respective contribution of exogenous and intramitochondrially formed ATP to D-glucose phosphorylation by mitochondria-bound hexokinase was examined in both rat liver and pancreatic islet mitochondria by comparing the generation of D-glucose 6-[32P]phosphate from exogenous [gamma-32P]ATP to the total rate of D-[U-14C]glucose phosphorylation. In liver mitochondria, the fractional contribution of exogenous ATP to D-glucose phosphorylation ranged from 4 to 74%, depending on the availability of endogenous ATP formed by either oxidative phosphorylation or in the reaction catalyzed by adenylate kinase. Likewise, in islet mitochondria exposed to exogenous ATP but deprived of exogenous nutrient, about 60% of D-glucose phosphorylation was supported by mitochondrial ATP. Such a fractional contribution was further increased in the presence of ADP and succinate, and suppressed by mitochondrial poisons. It is concluded that, in islet like in liver mitochondria, mitochondrial ATP is used preferentially to exogenous ATP as a substrate for D-glucose phosphorylation by mitochondria-bound hexokinase. This may favour the maintenance of a high cytosolic ATP concentration in glucose-stimulated islet cells.

Glucose turnover in hyperthyroid patients with normal glucose tolerance.

To determine the diabetogenic effect(s) of thyroid hormones, we simultaneously measured glucose turnover in six hyperthyroid patients and six normal subjects. All had normal fasting blood glucose concentration and oral glucose tolerance test values. We determined hepatic total glucose output (HTGO) and total glucose phosphorylation with [2-3H]glucose and hepatic glucose production (HGP) and irreversible glucose uptake using [6-3H]glucose. The difference between the two turnover rates indicates the extent of hepatic glucose cycling (glucose in equilibrium glucose-6-phosphate). Measurements were made both in the postabsorptive steady state and during a 2-h glucose infusion (11.1 mumol/kg.min). The postabsorptive HTGO and total glucose phosphorylation were increased in the hyperthyroid patients [13.5 +/- 0.8 (+/- SE) vs. 11.3 +/- 0.4 mumol/kg.min; P less than 0.05]. HGP and irreversible glucose uptake also were slightly but not significantly higher. During the glucose infusion, HTGO and HGP were less suppressed in the hyperthyroid patients than in the normal subjects, while the increments in peripheral glucose uptake were normal. In hyperthyroidism, glucose cycling was increased both postabsorptively (2.35 +/- 0.27 vs. 1.17 +/- 0.25 mumol/kg.min; P less than 0.025) and during glucose infusion (2.57 +/- 0.34 vs. 1.31 +/- 0.35 mumol/kg.min; P less than 0.05). We conclude that increases in HTGO and HGP are important features of hyperthyroidism, especially during glucose infusion. The increase in GC indicates increased activities of both glucokinase and glucose-6-phosphatase. The diabetogenic effect of hyperthyroidism, as revealed most markedly by [2-3H]glucose, could be accounted for by augmented glucose production, possibly due to increased glucose-6-phosphatase activity.