5-N-Substituted-2-(substituted benzenesulphonyl) glutamines as antitumor agents. Part II: synthesis, biological activity and QSAR study

Cancer is a major killer disease throughout human history. Thus, cancer becomes a major point of interest in life science. It was proved that cancer is a nitrogen trap and tumor cells are avid glutamine consumers. The non-essential amino acid glutamine, which is a glutamic acid derivative, supplies its amide nitrogen to tumor cells in the biosynthesis of purine and pyrimidine bases of nucleic acids as well as takes part in protein synthesis. Based on these and in continuation of our composite programme of development of new potential anticancer agents through rational drug design, 17 new 5-N-Substituted-2-(substituted benzenesulphonyl) glutamines were selected for synthesis. These compounds as well as 36 earlier synthesized glutamine analogues were screened for antitumor activity using percentage inhibition of tumor cell count as the activity parameter. QSAR study was performed with 53 compounds in order to design leads with increased effectiveness for antitumor activity using both physicochemical and topological parameters. QSAR study showed that steric effect on the aromatic ring is conducive to the activity. n-butyl substitution on aliphatic side chain and atom no 12 is important for antitumor activity of glutamine analogues.     

Pathways of the synthesis of glutamic acid by cephalosporin C-producing Acremonium chrysogenum

There were observed two pathways of glutamic acid formation in two strains of Acremonium chrysogenum differing in the production levels of cephalosporin C. The pathway involving glutamate dehydrogenase is known. The other pathway involved amination catalyzed by glutamine synthetase. Activity of both the enzymes during intensive synthesis of the antibiotic was higher in the highly productive strain. Under conditions of limited nitrogen content in the medium production of glutamate during the antibiotic biosynthesis depended on glutamine synthetase. When there was an excess of nitrogen in the medium the main role in production of glutamic acid at the phase of cephalosporin synthesis was played by the other enzyme i. e. glutamate dehydrogenase. By the dynamics the curve of the glutamate dehydrogenase activity correlated with that of the antibiotic production.     

Structure-activity relationship of daptomycin analogues with substitution at (2S, 3R) 3-methyl glutamic acid position

Daptomycin is a highly effective lipopeptide antibiotic against Gram-positive pathogens. The presence of (2S, 3R) 3-methyl glutamic acid (mGlu) in daptomycin has been found to be important to the antibacterial activity. However the role of (2S, 3R) mGlu is yet to be revealed. Herein, we reported the syntheses of three daptomycin analogues with (2S, 3R) mGlu substituted by (2S, 3R) methyl glutamine (mGln), dimethyl glutamic acid and (2S, 3R) ethyl glutamic acid (eGlu), respectively, and their antibacterial activities. The detailed synthesis of dimethyl glutamic acid was also reported.     

On the synthesis of glutamine before and after fertilization of the sea urchin, Strongylocentrotus purpuratus

Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, have a much lower capacity for glutamine synthesis than do fertilized eggs. This difference is not caused by an alteration of glutamine synthetase activity attendant upon fertilization. Neither the specific activity of glutamine synthetase nor its pattern of activation by divalent metal ions is affected by fertilization. The enzyme from both fertilized and unfertilized eggs is activated by α-ketoglutarate and inhibited by ultimate end products of glutamine metabolism. This type of regulation is similar to that seen with many other eucaryotic glutamine synthetases.Unfertilized eggs take up less glutamic acid than do fertilized eggs when the amino acid is presented at high concentrations (12.5 mM), whereas there is no difference in glutamic acid uptake at low concentrations (5 μM). Under conditions where glutamate uptake is identical, unfertilized eggs are dependent upon exogenous ammonia for glutamine synthesis in vivo; fertilized eggs are able to synthesize glutamine in the absence of added ammonia. Thus, our data suggest that the increased capacity for glutamine synthesis after fertilization is related to an increased availability of the substrate, ammonia.     

ACTINOMYCIN-D ENHANCEMENT OF GLUTAMINE SYNTHETASE ACTIVITY IN CHICK EMBRYO RETINAS CULTURED IN THE PRESENCE OF CORTISOL

Cortisol can prematurely induce glutamine synthetase activity in the chick embryo retina. Under appropriate conditions, this effect can be enhanced by addition of low levels of actinomycin D; this enhancement is reversibly inhibited by cycloheximide. The magnitude of the effect is a function of time of exposure to hormone as well as antibiotic and is also a function of the age of the embryo; within the limits of the present study it did not appear to be a function of actinomycin-D concentration. The data are discussed in terms of current ideas of possible control mechanisms in animal cells.     

INFLUENCE OF AMMONIUM AND NITRATE ON THE PROTEIN- AND FREE AMINO ACIDS IN SHOOTS OF WHEAT SEEDLINGS

Weissman , Gerard S. (Rutgers U., Camden, N. J.) Influence of ammonium and nitrate on the protein- and amino acids in shoots of wheat seedlings. Amer. Jour. Bot. 46(5): 339–346. 1959.—Total and protein nitrogen per shoot of wheat seedlings grown with endosperm attached increased at a steady rate during a 96-hr. growth period, and protein nitrogen, as a percentage of total nitrogen, remained constant at about 53%. Total and protein nitrogen concentration was greatest for 24-hr. shoots and declined as the shoots became older. Total and protein nitrogen were determined in 96-hr. shoots of seedlings grown with endosperm attached but also supplied with ammonium, nitrate, or both in the culture solution. Total nitrogen was greatest in shoots supplied with ammonium, but only 38% was in the form of protein. Maximum protein synthesis occurred in shoots grown in both ammonium and nitrate and protein nitrogen as a percentage of total nitrogen approximated that achieved in shoots lacking nitrogen in the culture solution. The protein amino acid composition of 48-, 72-, and 96-hr. shoots was very similar but differed from 24-hr. shoots which contained higher percentages of arginine and lysine and lower percentages of alanine and threonine. This may be correlated with the higher proportion of meristematic cells in 24-hr. shoots. The protein amino acids in shoots grown with ammonium resembled that of shoots lacking nitrogen in the culture solution, but nitrate shoot protein contained a higher percentage of arginine and a lower percentage of lysine. Nitrate may stimulate the formation of enzymes, possibly of a nitrate-reducing system, with high arginine- low lysine content. Free asparagine and glutamine were both at a maximum in ammonium shoots and at a minimum in nitrate shoots, but asparagine predominated in shoots supplied with ammonium while glutamine was greatest in nitrate shoots. Aspartic acid, asparagine, and glutamine appeared to have ammonia-storage functions, but glutamic acid appeared to be primarily concerned with protein synthesis. Amino acid accumulation was greatest in shoots supplied with both ammonium and nitrate. Protein synthesis in these appeared to be limited by inadequate concentrations of glutamic acid and proline. A hypothesis is proposed in explanation of the high glutamic acid concentration in shoots provided with ammonium and nitrate.     

METABOLISM OF GLUTAMIC ACID AND RELATED AMINO ACIDS IN THE BRAIN STUDIED WITH 14C-LABELLED GLUCOSE, BUTYRIC ACID AND GLUTAMIC ACID IN HYPERCAPNIC RATS

Abstract— The influence of hypercapnia on the metabolism of glutamic acid, aspartic acid, glutamine and GABA in rat brain was studied using three different precursors. Acute hypercapnia induced a fall in the concentration of glutamic and aspartic acid, and a rise in the concentration of glutamine and GABA. Acute hypercapnia had a profound effect on the relative specific radioactivity of glutamine indicating that the excess glutamine, present in the brain in hypercapnia, was synthetized from glutamic acid in the compartment where it could become quickly labelled from butyric and glutamic acid, but not from glucose. This effect was maintained in chronic hypercapnia.     

Distinct intracellular localization of Gpd1p and Gpd2p, the two yeast isoforms of NAD+-dependent glycerol-3-phosphate dehydrogenase, explains their different contributions to redox-driven glycerol production

During anaerobiosis Saccharomyces cerevisiae strongly increases glycerol production to provide for non-respiratory oxidation of NADH to NAD(+). We here report that respiratory-deficient cells become strictly dependent on the Gpd2p isoform of the NAD(+)-linked glycerol-3-phosphate dehydrogenase (Gpd). The growth inhibition of respiratory incompetent cox18Delta cells lacking GPD2 is reversed by the addition of acetoin, an alternative sink for NADH oxidation. Growth is also restored by addition of lysine or glutamic acid/glutamine, the synthesis of which involves production of mitochondrial NADH. Lysine produced a stronger growth stimulating effect than glutamic acid consistent with an upregulated expression of the IDP3 gene for peroxisomal synthesis of the glutamate precursor alpha-ketoglutarate. Gpd2p is known to be a cytosolic protein but possesses a classical mitochondrial presequence, which we show is sufficient for mitochondrial targeting. A partial mitochondrial localization of Gpd2p will provide for establishment of intramitochondrial redox balance under non-respiratory conditions. Gpd1p, the other Gpd isoform, is partly cytosolic and partly peroxisomal and becomes more strictly peroxisomal in respiratory-deficient mutants. The different cellular distribution of Gpd1p and Gpd2p thus appears to be the main reason Gpd1p cannot substitute for Gpd2p in cox18Deltagpd2Delta cells, despite similar kinetic characteristics of the two iso-enzymes.     

Incorporation of radioactivity from amino acids in the presence of cycloheximide demonstrates gluconeogenic activity in Mucor rouxii.

Mucor rouxii cells induced for gluconeogenesis incorporated radioactivity from [14C]glutamic acid into trichloroacetic acid-precipitable material in the presence of 200 micrograms of cycloheximide per ml. This metabolic capacity was repressed by hexoses and required amino acids for induction. These results suggest that the incorporation of amino acids in the presence of cycloheximide represents gluconeogenic activity with associated polysaccharide synthesis.     

INTRACELLULAR AMINO ACID CONTENT OF NEURONAL, GLIAL, AND NON-NEURAL CELL CULTURES: THE RELATIONSHIP TO GLUTAMIC ACID COMPARTMENTATION

Abstract— A protocol for the accurate determination of intracellular levels of amino acids in tissue cultured cells has been developed and used in the measurement of intracellular amino acids levels in neuronal, glial, and non-neural cell lines, with the objective of establishing morphological correlates for large and small glutamic acid compartments and of examining hypotheses for the morphological basis of glutamic acid compartmentation. This survey of intracellular amino acid levels has revealed striking differences among the cell lines tested, but these differences did not correlate with cell type, i.e. neuronal vs glial, in contrast to earlier results (R ose , 1968) based on bulk separated neuronal and pial fractions from rat brain. Amino acid levels were found to be dependent upon tissue culture conditions, yet reproducible differences could be observed when growth and experimental conditions were carefully controlled. Glutamic acid levels for various cell lines ranged from 50.8 ± 14.3 to 158 ± 8.5 nmol/mg protein. Intracellular glutamine levels demonstrated even greater difference, with values ranging from 0.8 ± 0.2 to 107 ± 42.4 nmol/mg protein. Statistically significant differences in intracellular amino acid levels between cell lines were also observed for aspartic acid, praline, glycine, alanine, valine, cystathionine, isoleucine, and leucine. A number of cell lines demonstrating highly elevated elevated levels of γ-aminobutyrate and β-alanine were identified. The significance of neuronal and glial levels of glutamic acid, glutamine and γ-aminobutyrate to models for glutamic acid compartmentation is discussed.     

Glutathione stimulates A549 cell proliferation in glutamine-deficient culture: The effect of glutamate supplementation

Extracellular glutathione (GSH) is degraded by an external cell-surface enzyme, γ-glutamyltranspeptidase (γ-GT). The products are transported into cells to participate in important cellular processes. In the present study, we tested the hypothesis that extracellular GSH is a source of glutamic acid for cells that express γ-GT. Under a glutamine-deficient culture condition, the extracellular GSH-supplemented glutamic acid would enhance intracellular glutamine synthesis, thereby stimulating cell proliferation. Human lung carcinoma A549 cells were cultured in glutamine-deficient Dulbecco's modified Eagle medium, and they did not proliferate unless glutamine was supplemented. Extracellular GSH, however, provoked a partial proliferation. The GSH effect correlated with a high level of γ-GT activity and an increased intracellular level of glutamic acid. A constituent amino acid of GSH, glutamic acid but not cysteine, produced the same growth-stimulatory effect as GSH. Furthermore, neither oxothiazolidine-4-carboxylate (OTC), a celluar cysteine-delivery compound, nor cysteinylglycine, a dipeptide released from the γ-GT reaction, stimulated cell proliferation. Moreover, buthionine sulfoximine (BSO), a selective inhibitor of γ-glutamylcysteine synthetase, enhanced the GSH growth stimulatory effect, suggesting that increased cellular GSH synthesis does not correlate with cell growth stimulation. The results obtained demonstrated that glutamine is required for A549 cell proliferation and exogenous GSH partially substitutes for the growth stimulatory action of glutamine. It also suggests that the glutamic acid rather than the cysteine released from the GSH is responsible for the cell proliferation. © 1994 Wiley-Liss, Inc.     

Ribonucleic acid synthesis in yeast. The effect of cycloheximide on the synthesis of ribonucleic acid in Saccharomyces carlsbergensis

1. Cycloheximide causes the release of the control amino acids have over RNA synthesis in Saccharomyces carlsbergensis N.C.T.C. 74. 2. The antibiotic causes a gradual deceleration of RNA formation. After incubation for 60min. at 30 degrees RNA synthesis usually proceeds at a rate only a few per cent of that of the untreated control. 3. In the presence of cycloheximide two types of RNA accumulate in the cell: soluble RNA and a high-molecular-weight RNA. The latter has a base composition intermediate between those of yeast DNA and yeast ribosomal RNA, and sediments in a sucrose gradient at a rate faster than that of the 23s ribosomal RNA component. 4. Yeast ribosomal RNA contains methylated bases. Judged from the incorporation of [Me-(14)C]methionine, the extent of methylation of ribosomal RNA is about 20% of that of the ;soluble' RNA fraction. The high-molecular-weight RNA formed in the presence of cycloheximide is less methylated than normal RNA. In this case the sucrose-density-gradient sedimentation patterns of newly methylated and newly synthesized RNA do not coincide. 5. In the presence of cycloheximide, polysomal material accumulates, indicating that messenger RNA is formed. 6. The effect of the antibiotic on protein and RNA synthesis can be abolished by washing of the cells. The RNA that has accumulated during incubation of the cells with the antibiotic is not stable on removal of cycloheximide. 7. The results presented in this study are discussed in relation to the regulation of RNA formation in yeast.     

Studies on the biosynthesis of iturin, an antibiotic of Bacillus subtilis, and a lipopeptide containing beta-hydroxy fatty acids

The biosynthesis of iturin, an antibiotic containing a beta-amino fatty acid, was studied by incubating Bacillus subtilis in the presence of various 14C-labelled precursors. Sodium acetate or palmitic acid were incorporated into the beta-amino acids of iturin. Among the alpha-amino acids (asparagine, glutamine, serine, proline and tyrosine) in the peptidic part of iturin, asparagine appears to be the best precursor. In the presence of sodium [14C]acetate or [14C]asparagine, there was a synthesis of radioactive compound (compound X) before the synthesis of radioactive iturin. Compound X contained asparagine and/or aspartic acid, glutamine and/or glutamic acid and beta-hydroxy fatty acids.     

Regulation of glutamine synthetase and glutaminase activities in cultured skeletal muscle cells

Glutamine is synthesized in skeletal muscle, released to the circulation, and transported to other tissues, where it may provide important substrate for gluconeogenesis, ammoniagenesis, and energy-yielding pathways. With the ultimate goal of delineating the factors that control glutamine production and release by skeletal muscle, we have studied the regulation of two key enzymes, glutamine synthetase and glutaminase, in the L6 line of rat skeletal muscle cells grown in monolayer culture. The cultured myotubes were found to have glutamine synthetase and phosphate-dependent glutaminase activities. Glutamine synthetase activity was increased following incubation (1) in glutamine-free medium (threefold); (2) in medium containing high glutamic acid concentrations (fourfold); and (3) in medium supplemented with dexamethasone (threefold). In each case the increase in glutamine synthetase activity required several hours to reach a maximum and was prevented by cycloheximide, suggesting that the change occurred through increased enzyme biosynthesis. No substances tested were found to affect glutaminase activity. We conclude that glutamine synthetase in cultured skeletal muscle is responsive to substrate, product, and hormonal regulation.     

ANISOMYCIN,ACETOXYCYCLOHEXIMIDE, CYCLOHEXIMIDE,AND PUROMYCIN AS INHIBITORS OF RAT BRAIN ACETYLCHOLINESTERASE IN VITRO1

A kinetic analysis of the interaction of anisomycin, acetoxycycloheximide, cycloheximide, and puromycin with acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) in rat brain homogenate shows that all of these protein synthesis inhibitors are also inhibitors or this enzyme. Puromycitl aminonucleoside, a puromycin analog without antibiotic activity, was also found to be an inhibitor of acetylcholinesterase activity much like puromycin. Anisomycin appeared to be a competitive inhibitor whereas all of the other compounds showed mixed inhibition. The apparent 10.5 values for inhibition of rat brain acetylcholinesterase at 50 μM substrate were: anisomycin, 3 mM; acetoxycycloheximide, 1 mM; cycloheximide, 2.2 mM; puromycin, 0.5 mM and puromycin aminonucleoside, 0.6 mM.     

End products of glucose and glutamine metabolism by L929 cells

Products of glucose and glutamine metabolism by L929 cells were detected and quantitated by gas chromatography and mass spectrometry of the oxime-trimethylsilyl derivatives. This method allowed detection and identification of all major carboxylic and amino acids produced in the system. Although lactic acid was expected to be the major product, alanine, citric, glutamic, aspartic, and pyruvic acids were also released into the culture medium at significant rates. Incorporation of labeled carbon from D-[U-13C]glucose showed that the alanine, lactic, and pyruvic acids were derived from glucose as was one-third of the citric acid carbon. The rate of glucose utilization for production of these end products was 29-fold greater than the rate of glucose oxidation to CO2, and calculated ATP production from alanine and pyruvate synthesis exceeded that from lactate synthesis by nearly 2-fold. Utilization of glutamine for synthesis of aspartic, glutamic, and citric acids also exceeded the rate of glutamine oxidation, thereby making end-product synthesis from glucose and glutamine the dominant cellular metabolic activity. In the absence of glucose, synthesis and intracellular levels of aspartic and glutamic acids increased, whereas synthesis and cell content of the other acids decreased markedly. This response is consistent with the metabolic pattern proposed by Moreadith and Lehninger (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221) in which much of the glutamine used by these cells is converted to aspartate in the absence of a pyruvate source and to aspartate or citrate in the presence of pyruvate.     

OBSERVATIONS ON THE ACID PHOSPHATASES OF EUGLENA GRACILIS

When a bleached strain of Euglena is maintained in a medium containing very low con centrations of phosphate, the acid phosphatase activity increases. The increase in acid phosphatase activity is prevented by Actinomycin D and by p-fluorophenylalanine (PFA), indicating that the increased activity is due to de novo synthesis of acid phosphatase. When phosphate is replenished, the acid phosphatase activity decreases to the level characteristic of uninduced cells before there is any appreciable cell division. When cell division resumes in the presence of PFA, the level of acid phosphatase activity remains approximately constant. This indicates that there are two different phosphatases: a constitutive enzyme, whose synthesis is insensitive to the presence of PFA, and an induced enzyme, whose synthesis is sensitive to PFA. These enzymes are not equally sensitive to changes in pH and in fluoride concentration, thus permitting them to be assayed individually in whole toluene-treated cells. Induced cells also acquire the ability to remove phosphate from the medium very rapidly.     

The effect of cycloheximide on Euglena gracilis phenylalanyl-tRNA synthetases

The effect of cycloheximide on the chloroplastic, cytoplasmic and mitochondrial phenylalanyltransferRNA synthetases of Euglena gracilis was studied by growing both logarithmic and stationary phase cultures in the presence of the antibiotic. Enzyme activity was measured relative to untreated control cultures. At very low concentrations of cycloheximide (1 g/ml), all three log phase enzymes showed an increase in activity of 40–50%. At slightly higher concentrations (2.5 g/ml), the phenylalanyl-tRNA synthetase activities were comparable to those of the control cultures. At a cycloheximide concentration of 5g/ml the enzyme activities from stationary phase cultures showed only very slight decreases (5–20%). The cytoplasmic and mitochondrial enzymes behaved similarly in log phase cultures at this concentration. However, the chloroplastic phenylalanyl-tRNA synthetase from log phase cultures treated with 5g/ml cycloheximide showed a marked decrease in activity (70%). A further increase in antibiotic concentration to 10g/ml resulted in significant losses of activity of all three enzymes, from both growth stages. The implications of the data with regard to identification of the site(s) of chloroplast enzyme synthesis are discussed.     

Effect of amino acids on exoprotease synthesis by Bacillus thuringiensis

The influence of certain L-amino acids and their mixtures on the synthesis of exoprotease from Bacillus thuringiensis was studied. Physiological experiments showed that the mixture of 20 amino acids added to the artificial medium repressed the synthesis of exoprotease. Among the compounds studied there are both the compounds which stimulate the synthesis of exoprotease (glutamic and aspartic acids, glycine), and the compounds which repress the synthesis of the enzyme (proline, tryptophane, tyrosine, asparagine, serine, cystein). None of the amino acids caused a change in the exoprotease activity. It has been assumed that the repression of the protease synthesis in the presence of the amino acids is accomplished by ammonium ions, which are formed when using the amino acids of Bac. thuringiensis. The glutamine synthetase activity of cells was determined during the growth of Bac. thuringiensis both on a medium containing triptone and after the addition of certain amino acids to the cell suspension. The correlation between the influence of different amino acids on the synthesis of exoprotease and the glutamine synthetase activity was demonstrated.     

THE REGENERATION OF CILIA IN PARTIALLY DECILIATED TETRAHYMENA

Partial deciliation of Tetrahymena resulted in cells losing 75% of their cilia, with the balance being paralyzed. The paralyzed cilia are resorbed in the first 20 min after partial deciliation, and regeneration of cilia begins before resorption is completed. Inhibition of protein synthesis with cycloheximide does not inhibit ciliary resorption or regeneration, whereas vinblastine sulfate inhibits regeneration but not resorption. Inhibition of regeneration occurs in completely deciliated cells when they are treated with cyclohexmimide or vinblastine sulfate. It is concluded that the resorbing cilia contribute materials which allow regeneration to occur in the absence of protein synthesis. The volume of cilia regenerated in the presence of cycloheximide in partially deciliated cells is greater than the ciliary volume which is resorbed. This suggests the Tetrahymena cells have a pool of ciliary precursors. This pool does not contribute materials for regeneration in completely deciliated cells which are treated with cycloheximide. It is concluded that resorbing cilia in partially deciliated cells contribute materials which potentiate assembly of cilia from the pool of precursors.