Two-electron reduction of quinones by Enterobacter cloacae NAD(P)H:nitroreductase: quantitative structure-activity relationships

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes two-electron reduction of a series of quinoidal compounds according to a "ping-pong" scheme, with marked substrate inhibition by quinones. The steady-state catalytic constants (k(cat)) range from 0.1 to 1600s(-1), and bimolecular rate constants (k(cat)/K(m)) range from 10(3) to 10(8)M(-1)s(-1). Quinones, nitroaromatic compounds and competitive to NADH inhibitor dicumarol, quench the flavin mononucleotide (FMN) fluorescence of nitroreductase. The reactivity of NR with single-electron acceptors is consistent with an "outer-sphere" electron transfer model, taking into account high potential of FMN semiquinone/FMNH(-) couple and good solvent accessibility of FMN. However, the single-electron acceptor 1,1(')-dibenzyl-4,4(')-bipyridinium was far less reactive than quinones possessing similar single-electron reduction potentials (E(1)(7)). For all quinoidal compounds except 2-hydroxy-1,4-naphthoquinones, there existed parabolic correlations between the log of rate constants of quinone reduction and their E(1)(7) or hydride-transfer potential (E(7)(Q/QH(-))). Based on pH dependence of rate constants, a single-step hydride transfer seems to be a more feasible quinone reduction mechanism. The reactivities of 2-hydroxy-1,4-naphthoquinones were much higher than expected from their reduction potential. Most probably, their enhanced reactivity was determined by their binding at or close to the binding site of NADH and dicumarol, whereas other quinones used the alternative, currently unidentified binding site.     

Effects of some analgesic anaesthetic drugs on human erythrocyte glutathione reductase: an in vitro study

Inhibitory effects of some analgesic and anaesthetic drugs on human erythrocyte glutathione reductase were investigated. For this purpose, human erythrocyte glutathione reductase was initially purified 2139-fold in a yield of 29% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis confirmed the purity of the enzyme by sharing a single band. A constant temperature (+4 degrees C) was maintained during the purification process. Diclofenac sodium, ketoprofen, lornoxicam, tenoxicam, etomidate, morphine and propofol exhibited inhibitory effects on the enzyme in vitro using the Beutler assay method. K(i) constants and IC(50) values for drugs were determined from Lineweaver-Burk graphs and plotting activity % versus [I] graphs, respectively. The IC(50) values of diclofenac sodium, ketoprofen, lornoxicam, propofol, tenoxicam, etomidate and morphine were 7.265, 6.278, 0.3, 0.242, 0.082, 0.0523 and 0.0128 mM and the K(i) constants were 23.97 +/- 2.1, 22.14 +/- 7.6, 0.42 +/- 0.18, 0.418 +/- 0.056, 0.13 +/- 0.025, 0.0725 +/- 0.0029 and 0.0165 +/- 0.0013 mM, respectively. While diclofenac sodium, ketoprofen, lornoxicam, tenoxicam etomidate and morphine showed competitive inhibition, propofol displayed noncompetitive inhibition.     

Inhibition of glutathione reductase activity by a carbamoylating nitrosourea: effect on cellular radiosensitivity.

Nitrosoureas inactivate cellular glutathione reductase. N,'N'1,3-bis(trans-4-hydroxycyclohexyl)-N'-nitrosoureas (BCyNU), a nitrosourea reported to selectively inhibit glutathione reductase (GR) activity, was examined to determine if it could be used as a means to inhibit cellular levels of this enzyme in radiobiology studies. Confirmation of drug-induced inhibition of GR activity was demonstrated using a cell-free model system employing purified GR. Cellular studies with Chinese hamster V79A03 showed that BCyNU decreased cellular glutathione content concomitant with an inhibition of specific GR activity. Under relatively nontoxic conditions, cellular exposure to BCyNU (25 microM, 0.25 h) either before or after radiation treatment, increased cellular radiosensitivity with the optimum time for drug addition being immediately following radiation. At a BCyNU dosage which produced less than or equal to 5% cell toxicity, a marked decrease in radioresistance was characterized as a reduction in both Dq (24 +/- 1.5%) and Do (8 +/- 0.5%) concomitant with a 25 +/- 2% decrease in cellular glutathione reductase (GR) activity. At cytotoxic drug dosages (25 microM, 1 h; cell survival 79 +/- 7%), a marked radiosensitization manifested by a 1.25 +/- .07-fold reduction in the Dq was observed concomitant with a 49 +/- 4% decrease in GR activity. Using cells enriched in different stages of the cell cycle, BCyNU caused cell-age dependent cytotoxicity with preferential killing of cells in the radioresistant late-S-phase, a likely explanation for its radiosensitizing capabilities at high drug dosages. Data obtained at nontoxic drug dosages suggest that GR-inactivation may be an important component of cellular response to free-radical induced damage.     

Role of single-electron oxidation potential and lipophilicity in the antiplasmodial in vitro activity of polyphenols: comparison to mammalian cells

In spite of extensive studies, the structure-activity relationships in the action of polyphenols against the malaria parasite Plasmodium falciparum are poorly understood so far. As the mammalian cell cytotoxicity of polyphenols shows a negative dependence on the potential of the phenoxyl radical/phenol redox couple (E(2)(7)), due to the involvement of prooxidant events, and a positive dependence on the octanol/water distribution coefficient at pH 7.0 (log D), we examined the role of these parameters in their antiplasmodial in vitro activity. We found that the concentrations of hydroxybenzenes causing 50% inhibition of the growth of P falciparum strain FcB1 (IC50) are described by the regression log IC50 (microM) = 0.36 + 1.81 E(2)(7) (V) - 0.10 log D [n = 11, r2 = 0.760, F(2.8) = 12.03]. The IC50 values of flavonoids (n = 5), comprising a separate less active series, did not depend on their E(2)(7) values, 0.33 V-0.75 V. These findings were similar to the mammalian cell cytotoxicity data. However, the mammalian cell cytotoxicity of hydroxybenzenes showed more pronounced dependence on their E(2)(7) values [delta log CL50/delta E(2)(7) = (6.9 - 5.1) V(-1), where CL50 is the compound concentration for 50% cell survival] than on their antiplasmodial activity. Although it is unclear whether the prooxidant action is the main factor in the antiplasmodial action of polyphenols or not, our data showed that the ease of their oxidation (decrease in E(2)(7)) may enhance their activity. On the other hand, the different sensitivity of the mammalian cell cytotoxicity and the antiplasmodial activity of the hydroxybenzenes to their E(2)(7) values implied that compounds with high oxidation potential may be used as relatively efficient antiplasmodial agents with low mammalian cell cytotoxicity.     

Slow-binding inhibition of human prostaglandin endoperoxide synthase-2 with darbufelone, an isoform-selective antiinflammatory di-tert-butyl phenol.

The antiinflammatory agent darbufelone, ((Z)-5-[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl] methylene]-2-imino-4-thiazolidinone, methanesulfonate salt), was discovered as a dual inhibitor of cellular prostaglandin and leukotriene production. To study the mechanism of action of this drug, we expressed human prostaglandin endoperoxide synthase-1 (PGHS-1) and PGHS-2 and purified the recombinant enzymes using buffers that contain octylglucoside. In cyclooxygenase assays following a 15-min incubation of enzyme with inhibitor, darbufelone potently inhibits PGHS-2 (IC(50) = 0.19 microM) but is much less potent with PGHS-1 (IC(50) = 20 microM). Interestingly, when the assay buffer contains traces of Tween 20 (0.0001%), darbufelone appears inactive with PGHS-2 due to a detergent interaction that is detectable by absorption spectroscopy. We therefore used octylglucoside, which does not affect darbufelone in this way, in place of Tween 20 in our PGHS buffers. Inhibition of PGHS-2 with darbufelone is time dependent: with no preincubation, darbufelone is a weak inhibitor (IC(50) = 14 microM), but after a 30-min incubation it is 20-fold more potent. Plots of PGHS-2 activity vs preincubation time at various darbufelone concentrations reach a plateau. This finding is inconsistent with irreversible or one-step slow-binding inhibition. A two-step slow-binding inhibition model is proposed in which the E.I complex (K(i) = 6.2 +/- 1.9 to 14 +/- 1 microM) slowly transforms (k(5) = 0.015-0.030 s(-)(1)) to a tightly bound E.I form with K(i) = 0.63 +/- 0.07 microM and k(6) = 0.0034 s(-)(1). In steady-state kinetics inhibition experiments performed with no preincubation, we find that darbufelone is a noncompetitive inhibitor of PGHS-2 (K(i) = 10 +/- 5 microM). Darbufelone quenches the fluorescence of PGHS-2 at 325 nm (lambda(ex) = 280 nm) with K(d) = 0.98 +/- 0.03 microM. The PGHS substrate, arachidonate, and various cyclooxygenase inhibitors do not alter this binding affinity of darbufelone but a structural analogue of darbufelone competes directly for binding to PGHS-2. Di-tert-butyl phenols such as darbufelone may inhibit PGHS-2 by exploiting a previously unrecognized binding site on the enzyme.     

Marine natural products from the Turkish sponge Agelas oroides that inhibit the enoyl reductases from Plasmodium falciparum, Mycobacterium tuberculosis and Escherichia coli

The type II fatty acid pathway (FAS-II) is a validated target for antimicrobial drug discovery. An activity-guided isolation procedure based on Plasmodium falciparum enoyl-ACP reductase (PfFabI) enzyme inhibition assay on the n-hexane-, the CHCl(3-) and the aq MeOH extracts of the Turkish marine sponge Agelas oroides yielded six pure metabolites [24-ethyl-cholest-5alpha-7-en-3-beta-ol (1), 4,5-dibromopyrrole-2-carboxylic acid methyl ester (2), 4,5-dibromopyrrole-2-carboxylic acid (3), (E)-oroidin (4), 3-amino-1-(2-aminoimidazoyl)-prop-1-ene (5), taurine (6)] and some minor, complex fatty acid mixtures (FAMA-FAMG). FAMA, consisting of a 1:2 mixture of (5Z,9Z)-5,9-tricosadienoic (7) and (5Z,9Z)-5,9-tetracosadienoic (8) acids, and FAMB composed of 8, (5Z,9Z)-5,9-pentacosadienoic (9) and (5Z,9Z)-5,9-hexacosadienoic (10) acids in approximately 3:3:2 ratio were the most active PfFabI inhibitory principles of the hexane extract (IC(50) values 0.35 microg/ml). (E)-Oroidin isolated as free base (4a) was identified as the active component of the CHCl(3) extract. Compound 4a was a more potent PfFabI inhibitor (IC(50) 0.30 microg/ml=0.77 microM) than the (E)-oroidin TFA salt (4b), the active and major component of the aq MeOH extract (IC(50) 5.0 microg/ml). Enzyme kinetic studies showed 4a to be an uncompetitive PfFabI inhibitor (K(i): 0.4+/-0.2 and 0.8+/-0.2 microM with respect to substrate and cofactor). In addition, FAMA and FAMD (mainly consisting of methyl-branched fatty acids) inhibited FabI of Mycobacterium tuberculosis (MtFabI, IC(50)s 9.4 and 8.2 microg/ml, respectively) and Escherichia coli (EcFabI, IC(50)s 0.5 and 0.07 microg/ml, respectively). The majority of the compounds exhibited in vitro antiplasmodial, as well as trypanocidal and leishmanicidal activities without cytotoxicity towards mammalian cells. This study represents the first marine metabolites that inhibit FabI, a clinically relevant enzyme target from the FAS-II pathway of several pathogenic microorganisms.     

The use of natural product scaffolds as leads in the search for trypanothione reductase inhibitors

Twenty-three heterocyclic compounds were evaluated for their potential as trypanothione reductase inhibitors. As a result, the harmaline, 10-thiaisoalloxazine, and aspidospermine frameworks were identified as the basis of inhibitors of Trypanosoma cruzi trypanothione reductase. Two new compounds showed moderately strong, linear competitive inhibition, namely N,N-dimethyl-N-[3-(7-methoxy-1-methyl-3,4-dihydro-9H-beta-carbolin-9-yl)propyl]amine (15) and 1,3-bis[3-(dimethylamino)propyl]-1,5-dihydro-2H-pyrimido[4,5-b][1,4]benzothiazine-2,4(3H)-dione (21), with K(i) values of 35.1+/-3.5microM and 26.9+/-1.9microM, respectively. Aspidospermine (25) inhibited T. cruzi TryR with a K(i) of 64.6+/-6.2microM. None of the compounds inhibited glutathione reductase. Their toxicity toward promastigotes of Leishmania amazonensis was assessed.     

Inhibitory effects of terpenoids from the fermented broth of the ascomycete Stilbohypoxylon elaeicola YMJ173 on nitric oxide production in RAW264.7 macrophages

A series of six isopimarane-type diterpene glycosides, along with an eremophilane-type sesquiterpene, i.e., elaeicolasides A-C (1-3, resp.), 16-(α-D-mannopyranosyloxy)isopimar-7-en-19-oic acid (4), hymatoxin K (5), hymatoxin L (6), and elaeicolalactone (7), were isolated from the AcOEt extract of the fermented broth of Stilbohypoxylon elaeicola YMJ173. Among these, 1-3 and 7 are new compounds based on their spectroscopic data and sugar composition analysis. The effects of 1-7 on the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells were evaluated. All these compounds inhibited NO production, detected as nitrite in the culture medium, in activated macrophages without any cytotoxicity at a concentration of 100?μM. Among these compounds, 2 showed a significant activity with the average maximum inhibition (E(max)) and median inhibitory concentration (IC(50)) values of 93.3±0.5% and 79.3±0.4?μM, respectively.     

Effects of some metal ions on human erythrocyte glutathione reductase: an in vitro study

In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg(2+), Cd(2+), Pb(2+), Cu(2+), Fe(3+) and Al3+ exhibited inhibitory effects on the enzyme in vitro. K(i) constants and IC(50) values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC(50) values of Pb(2+), Hg(2+), Cu(2+), Cd(2+), Fe(3+) and Al(3+) were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254+/-0.0027, 0.0378+/-0.0043, 0.0409+/-0.0048, 0.0558+/-0.0083, 0.403+/-0.043 and 1.137+/-0.2 mM, respectively. While Pb(2+), Hg(2+), Cd(2+) and Fe(3+) showed competitive inhibition, others displayed noncompetitive inhibition.     

Gold complexes inhibit mitochondrial thioredoxin reductase: consequences on mitochondrial functions

The effects of gold(I) complexes (auranofin, triethylphosphine gold and aurothiomalate), gold(III) complexes ([Au(2,2'-diethylendiamine)Cl]Cl(2), [(Au(2-(1,1-dimethylbenzyl)-pyridine) (CH(3)COO)(2)], [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine)(OH)](PF(6)), [Au(bipy(dmb)-H)(2,6-xylidine)](PF(6))), metal ions (zinc and cadmium acetate) and metal complexes (cisplatin, zinc pyrithione and tributyltin) on mitochondrial thioredoxin reductase and mitochondrial functions have been examined. Both gold(I) and gold(III) complexes are extremely efficient inhibitors of thioredoxin reductase showing IC(50) ranging from 0.020 to 1.42 microM while metal ions and complexes not containing gold are less effective, exhibiting IC(50) going from 11.8 to 76.0 microM. At variance with thioredoxin reductase, auranofin is completely ineffective in inhibiting glutathione peroxidase and glutathione reductase, while gold(III) compounds show some effect on glutathione peroxidase. The mitochondrial respiratory chain is scarcely affected by gold compounds while the other metal complexes and metal ions, in particular zinc ion and zinc pyrithione, show a more marked inhibitory effect that is reflected on a rapid induction of membrane potential decrease that precedes swelling. Therefore, differently from gold compounds, the various metal ions and metal complexes exert their effect on different targets indicating a lower specificity. It is concluded that gold compounds are highly specific inhibitors of mitochondrial thioredoxin reductase and this action influences other functions such as membrane permeability properties. Metal ions and metal complexes markedly inhibit the activity of thioredoxin reductase although to an extent lower than that of gold compounds. They also inhibit mitochondrial respiration, decrease membrane potential and, finally, induce swelling.     

Kinetic characterization of glutathione reductase from the malarial parasite Plasmodium falciparum. Comparison with the human enzyme

The homodimeric flavoenzyme glutathione reductase (GR) maintains high intracellular concentrations of the antioxidant glutathione (GSSG + NADPH + H(+) <--> 2 GSH + NADP(+)). Due to its central function in cellular redox metabolism, inhibition of GR from the malarial parasite Plasmodium falciparum represents an important approach to antimalarial drug development; therefore, the catalytic mechanism of GR from P. falciparum has been analyzed and compared with the human host enzyme. The reductive half-reaction is similar to the analogous reaction with GR from other species. The oxidative half-reaction is biphasic, reflecting formation and breakdown of a mixed disulfide between the interchange thiol and GSH. The equilibrium between the E(ox)-EH(2) and GSSG-GSH couples has been modeled showing that the Michaelis complex, mixed disulfide-GSH, is the predominant enzyme form as the oxidative half-reaction progresses; rate constants used in modeling allow calculation of an K(eq) from the Haldane relationship, 0.075, very similar to the K(eq) of the same reaction for the yeast enzyme (0.085) (Arscott, L. D., Veine, D. M., and Williams, C. H., Jr. (2000) Biochemistry 39, 4711-4721). Enzyme-monitored turnover indicates that E(FADH(-))(S-S). NADP(+) and E(FAD)(SH)(2).NADPH are dominant enzyme species in turnover. Since the individual forms of the enzyme differ in their susceptibility to inhibitors, the prevailing states of GR in the cell are of practical relevance.     

Effects of some antibiotics on human erythrocyte glutathione reductase: an in vitro study

Inhibitory effects of some antibiotics on purified human erythrocyte glutathione reductase were investigated. Human erythrocyte glutathione reductase was purified 2800-fold (29% yield) at 4 degrees C using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis showed a single band for the enzyme. Imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol, seftriaxon, vancomycin, cefuroxime and ornidazole exhibited inhibitory effects but clindamycin, lincomycin, amoxicillin, amikacin exhibited activatory effects on the enzyme in vitro. The IC(50) values of imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol, seftriaxon, vancomycin, cefuroxime and ornidazole were 0.030, 0.146, 0.59, 2.476, 2.36, 2.88, 4.83, 15.43 and 19.632 mM, respectively, and the K(i) constants were 0.06 +/- 0.01, 0.275 +/- 0.10, 0.85 +/- 0.05, 3.59 +/- 0.51, 3.85 +/- 0.40, 3.71 +/- 0.60, 15.11 +/- 2.50, 23.50 +/- 2.94 and 28.49 +/- 6.50 mM, respectively. While imipenem, rifamycin, sulfanylacetamide, ceftazidime, chloramphenicol and seftriaxon cefuroxime and ornidazole showed competitive inhibition, vankomycine displayed noncompetitive inhibition.     

Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity

Glutathione reductase (E C: 1.8.1.7; GR) was purified from rainbow trout (Oncorhynchus mykiss) liver, and some characteristics of the enzyme were investigated. The purification procedure consisted of four steps: preparation of homogenate, ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose-4B and gel filtration chromatography on Sephadex G-200. The enzyme, with a specific activity of 27.45 U/mg protein, was purified 1,654-fold with a yield of 41%. Optimal pH, stable pH, optimal temperature, optimum ionic strength, molecular mass, KM and Vmax values for GSSG and NADPH were also determined for the enzyme. In addition, Ki values and inhibition types were determined for GSH and NADP+. Additionally, inhibitory effects of metal ions (Cd+2, Cu+2, Pb+2, Hg+2, Fe+3 and Al+3) on glutathione reductase were investigated. Ki constants and IC50 values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I], respectively. IC50 values of Cd+2,Cu+2, Pb+2, Hg+2, Fe+3 and Al+3 were 0.0655, 0.082, 0.122, 0.509, 0.797 and 0.804 mM, and the Ki constants for Cd+2 and Cu+2 were 0.104+/-0.001, 0.117+/-0.001, respectively.     

The relation of glutathione reductase and diaphorase activity of glutathione reductase from Saccharomyces cerevisiae

Glutathione reductase from S. cerevisiae (EC 1.6.4.2) catalyzes the NADPH oxidation by glutathione in accordance with a "ping-pong" scheme. The catalytic constant kcat) is 240 s-1 (pH 7.0, 25 degrees C); kcat for the diaphorase reaction is 4-5 s-1. The enzyme activity does not change markedly at pH 5.5-8.0. At pH less than or equal to 7.0, NADP+ acts as a competitive inhibitor towards NADPH and as a noncompetitive inhibitor towards glutathione. NADP+ increases the diaphorase activity of the enzyme. The maximal activity is observed, when the NADP+/NADPH ratio exceeds 100. At pH 8.0, NADP+ acts as a mixed type inhibitor during the reduction of glutathione. High concentrations of NADP+ also inhibit the diaphorase activity due to the reoxidation of the reduced enzyme by NADP+ at pH 8.0. The redox potential of glutathione reductase calculated from the inhibition data is--306 mV (pH 8.0). Glutathione reductase reduces quinoidal compounds in an one-electron way. The hyperbolic dependence of the logarithm of the oxidation constant on the one electron reduction potential of quinone is observed. It is assumed that quinones oxidize the equilibtium fraction of the two-electron reduced enzyme containing reduced FAD.     

Effects of phenol compounds, glutathione analogues and a diuretic drug on glutathione S-transferase, glutathione reductase and glutathione peroxidase from canine erythrocytes.

1. Phenol compounds (ellagic acid, quercetin and purpurogallin), glutathione analogues (S-hexylglutathione and S-octylglutathione) and a diuretic drug (ethacrynic acid) were compared for their inhibitory effects on glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) in the canine erythrocytes. 2. All these compounds inhibited GST activity; quercetin was found to be the most potent inhibitor. 3. Ellagic acid, purpurogallin, quercetin and ethacrynic acid inhibited GR activity; S-hexylglutathione and S-octylglutathione had no effect on GR and GSH-Px activities. 4. Quercetin and purpurogallin inhibited GST non-competitively toward glutathione, whereas ellagic acid showed a competitive inhibition. Ellagic acid and purpurogallin inhibited GR non-competitively toward oxidized glutathione.     

Protective effect of unsymmetrical dichalcogenide, a novel antioxidant agent, in vitro and an in vivo model of brain oxidative damage

Unsymmetrical dichalcogenides, a class of organoselenium compounds, were screened for antioxidant activity in rat brain homogenates in vitro. Unsymmetrical dichalcogenides (1-3) were tested against lipid peroxidation induced by sodium nitroprusside (SNP) or malonate, and reactive species (RS) production induced by sodium azide in rat brain homogenates. Compounds 1 (without a substituent at the phenyl group), 2 (chloro substituent at the phenyl group bounded to the sulfur atom) and 3 (chloro substituent at the phenyl group bounded to the selenium atom) protected against lipid peroxidation induced by SNP. The IC50 values followed the order 3<2<1. Lipid peroxidation induced by malonate was also reduced by dichalcogenides 1, 2 and 3. The IC50 values were 3相似文献   

In vitro effects of compounds isolated from Sideritis brevibracteata on bovine kidney cortex glutathione reductase

Glutathione reductase (GR, E.C 1.6.4.2) is a flavoprotein that catalyzes NADPH-dependent reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH). The aim of this study was to investigate in vitro effects of phenolic compounds isolated from Sideritis brevibracteata on bovine kidney GR. The Sideritis species are widely found in nature and commonly used as medicinal plants. 7-O-glycosides of 8-OH-flavones (hypolaetin, isoscutellarein and 3'-hydroxy-4'-O-methylisoscutellarein) were isolated from aerial parts of Sideritis brevibracteata. These compounds inhibited bovine kidney cortex GR in a concentration-dependent manner. Kinetic characterization of the inhibition was also performed.     

In vitro effects of some drugs on human erythrocyte glutathione reductase

The effects of ketotifen, meloxicam, phenyramidol-HCl and gadopentetic acid on the enzyme activity of GR were studied using human erythrocyte glutathione reductase (GR) enzymes in vitro. The enzyme was purified 209-fold from human erythrocytes in a yield of 19% with 0.31?U/mg. The purification procedure involved the preparation of haemolysate, ammonium sulphate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies. In the in vitro studies, IC(50) values and K(i) constants were 0.012?mM and 0.0008?±?0.00021?mM for ketotifen; 0.029?mM and 0.0061?±?0.00127?mM for meloxicam; 0.99?mM and 0.4340?±?0.0890?mM for phenyramidol-HCl; 138?mM and 28.84?±?4.69?mM for gadopentetic acid, respectively, showing the inhibition effects on the purified enzyme. Phenyramidol-HCl showed competitive inhibition, whereas the others showed non-competitive inhibition.     

Inhibition of the human thioredoxin system. A molecular mechanism of mercury toxicity

Mercury toxicity mediated by different forms of mercury is a major health problem; however, the molecular mechanisms underlying toxicity remain elusive. We analyzed the effects of mercuric chloride (HgCl(2)) and monomethylmercury (MeHg) on the proteins of the mammalian thioredoxin system, thioredoxin reductase (TrxR) and thioredoxin (Trx), and of the glutaredoxin system, glutathione reductase (GR) and glutaredoxin (Grx). HgCl(2) and MeHg inhibited recombinant rat TrxR with IC(50) values of 7.2 and 19.7 nm, respectively. Fully reduced human Trx1 bound mercury and lost all five free thiols and activity after incubation with HgCl(2) or MeHg, but only HgCl(2) generated dimers. Mass spectra analysis demonstrated binding of 2.5 mol of Hg(2+) and 5 mol of MeHg(+)/mol of Trx1 with the very strong Hg(2+) complexes involving active site and structural disulfides. Inhibition of both TrxR and Trx activity was observed in HeLa and HEK 293 cells treated with HgCl(2) or MeHg. GR was inhibited by HgCl(2) and MeHg in vitro, but no decrease in GR activity was detected in cell extracts treated with mercurials. Human Grx1 showed similar reactivity as Trx1 with both mercurial compounds, with the loss of all free thiols and Grx dimerization in the presence of HgCl(2), but no inhibition of Grx activity was observed in lysates of HeLa cells exposed to mercury. Overall, mercury inhibition was selective toward the thioredoxin system. In particular, the remarkable potency of the mercury compounds to bind to the selenol-thiol in the active site of TrxR should be a major molecular mechanism of mercury toxicity.     

Oxazolones: new tyrosinase inhibitors; synthesis and their structure-activity relationships

The tyrosinase inhibitory potential of seventeen synthesized oxazolone derivatives has been evaluated and their structure-activity relationships developed in the present work. All the synthesized derivatives, 3-19, demonstrated excellent in vitro tyrosinase inhibitory properties having IC50 values in the range of 1.23+/-0.37-17.73+/-2.69 microM, whereas standard inhibitors l-mimosine and kojic acid have IC50 values 3.68+/-0.02 and 16.67+/-0.52 microM,, respectively. Compounds 4-8 having IC50 values 3.11+/-0.95, 3.51+/-0.25, 3.23+/-0.66, 1.23 +/- 0.37, and 2.15+/-0.75, respectively, were found to be very active members of the series, even better than both the standard inhibitors. However, compounds 3, 9-11, 13, 14, 16, 17, and 19 were found to be better than kojic acid but not l-mimosine. (2-Methyl-4-[E,2Z)-3-phenyl-2-propenyliden]-1,3-oxazol-5(4H)-one (7) bearing a cinnamyol residue at C-4 of oxazolone moiety and an IC50 = 1.23+/-0.37 microM was found to be the most active one among all tested compounds. These studies reveal that the substitution of functional group (s) at C-4 and C-2 positions plays a vital role in the activity of this series of compounds. It is concluded that compound 7 may act as a potential lead molecule to develop new drugs for the treatment of tyrosinase based disorders.