Inhibitors of hydroperoxide metabolism enhance ascorbate-induced cytotoxicity

Abstract

Pharmacological ascorbate, via its oxidation, has been proposed as a pro-drug for the delivery of H₂O₂ to tumors. Pharmacological ascorbate decreases clonogenic survival of pancreatic cancer cells, which can be reversed by treatment with scavengers of H₂O₂. The goal of this study was to determine if inhibitors of intracellular hydroperoxide detoxification could enhance the cytotoxic effects of ascorbate. Human pancreatic cancer cells were treated with ascorbate alone or in combination with inhibitors of hydroperoxide removal including the glutathione disulfide reductase inhibitor 1,3 bis (2-chloroethyl)-1-nitrosurea (BCNU), siRNA targeted to glutathione disulfide reductase (siGR), and 2-deoxy-D-glucose (2DG), which inhibits glucose metabolism. Changes in the intracellular concentration of H₂O₂ were determined by analysis of the rate of aminotriazole-mediated inactivation of endogenous catalase activity. Pharmacological ascorbate increased intracellular H₂O₂ and depleted intracellular glutathione. When inhibitors of H₂O₂ metabolism were combined with pharmacological ascorbate the increase in intracellular H₂O₂ was amplified and cytotoxicity was enhanced. We conclude that inclusion of agents that inhibit cellular peroxide removal produced by pharmacological ascorbate leads to changes in the intracellular redox state resulting in enhanced cytotoxicity.     

Detection of singlet (1O2) oxygen phosphorescence during chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide

Evidence for the production of singlet molecular oxygen (1O2) during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide has been obtained through the use of optical spectroscopy, oxygen electrode experiments, and electron spin resonance (ESR). ESR spin-trapping experiments with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) demonstrate the production of the ethyl peroxyl free radical during the chloroperoxidase/ethyl hydroperoxide reaction. Oxygen and acetaldehyde concentrations suggest that the production of ethyl peroxyl radicals constitutes less than 2% of the decomposition of ethyl hydroperoxide at the concentrations of reactants used. The phosphorescence of 1O2 at 1268 nm was observed during the chloroperoxidase-catalyzed decomposition of ethyl hydroperoxide in deuterium oxide buffer. Chloroperoxidase also catalyzes the decomposition of tert-butyl hydroperoxide to its corresponding peroxyl radical. Alkoxyl and alkyl-DMPO spin adducts were also detected. A much lower yield of 1O2 phosphorescence was observed during the chloroperoxidase-catalyzed decomposition of tert-butyl hydroperoxide. This phosphorescence probably arises through secondary production of alkyl peroxyl radicals. These results suggest that the initial enzyme-dependent production of ethyl peroxyl radicals is followed by enzyme-independent reaction of two peroxyl radicals through the tetroxide intermediate, as originally proposed by Russell (Russell, G. A. (1957) J. Am. Chem. Soc. 79, 3871-3877), to form acetaldehyde, ethyl alcohol, and molecular oxygen.     

Optimization of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)acetamide derivatives as arginine vasopressin V2 receptor agonists and discussion of their binding modes

A series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)acetamide derivatives were optimized to achieve potent agonistic activity, both in vitro and in vivo, for the arginine vasopressin V₂ receptor, resulting in the eventual discovery of compound 1g. Molecular modeling of compound 1g with V₂ receptor was also examined to evaluate the binding mode of this series of compounds.     

Localization of fatty acid hydroperoxide cleavage activity in membranes of cucumber fruit

The subcellular localization of the fatty acid hydroperoxide cleavage enzyme from cucumber fruit has been studied. Activity from the flesh tissue has been located in 3 fractions; plasma and Golgi membranes and endoplasmic reticulum, at equilibrium densities on sucrose gradients of 1.17, 1.15 and 1.12 g/cm³ respectively. Enzymatic activity and electron microscopy studies were carried out to identify plasma and Golgi membranes. Little activity was associated with microbodies (1.23), plastids (1.21) and mitochondria (1:19 g/cm³). However, chloroplasts isolated from the peel of the cucumber fruit contained a large amount of hydroperoxide cleavage activity.     

Tetrahydro-beta-carboline alkaloids that occur in foods and biological systems act as radical scavengers and antioxidants in the ABTS assay

Tetrahydro- β-carboline alkaloids that occur in foods such as wine, seasonings, vinegar and fruit products (juices, jams) acted as good radical scavengers (hydrogen- or electron donating) in the ABTS (2,2'-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay, and therefore, they could contribute to the beneficial antioxidant capacity attributed to foods. In contrast, the fully aromatic β-carbolines norharman and harman did not show any radical scavenger activity in the same assay. During the reaction with ABTS^.++ radical cation, tetrahydro- β-carboline-3-carboxylic acid such as 1-methyl-1,2,3,4-tetrahydro- β-carboline-3-carboxylic acid (MTCA) and 1-methyl-1,2,3,4-tetrahydro- β-carboline-1,3-dicarboxylic acid (MTCA-COOH) were converted to harman, whereas 1,2,3,4-tetrahydro- β-carboline-3-carboxylic acid (THCA) and 1,2,3,4-tetrahydro- β-carboline-1,3-dicarboxylic acid (THCA-COOH) afforded norharman. These results suggest that food and naturally-occurring tetrahydro- β-carboline alkaloids if accumulated in tissues, as reported elsewhere, might exhibit antioxidant activity.     

Tetrahydro-β-carboline Alkaloids that Occur in Foods and Biological Systems Act as Radical Scavengers and Antioxidants in the ABTS Assay

Tetrahydro- &#103 -carboline alkaloids that occur in foods such as wine, seasonings, vinegar and fruit products (juices, jams) acted as good radical scavengers (hydrogen- or electron donating) in the ABTS (2,2'-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay, and therefore, they could contribute to the beneficial antioxidant capacity attributed to foods. In contrast, the fully aromatic &#103 -carbolines norharman and harman did not show any radical scavenger activity in the same assay. During the reaction with ABTS^.++ radical cation, tetrahydro- &#103 -carboline-3-carboxylic acid such as 1-methyl-1,2,3,4-tetrahydro- &#103 -carboline-3-carboxylic acid (MTCA) and 1-methyl-1,2,3,4-tetrahydro- &#103 -carboline-1,3-dicarboxylic acid (MTCA-COOH) were converted to harman, whereas 1,2,3,4-tetrahydro- &#103 -carboline-3-carboxylic acid (THCA) and 1,2,3,4-tetrahydro- &#103 -carboline-1,3-dicarboxylic acid (THCA-COOH) afforded norharman. These results suggest that food and naturally-occurring tetrahydro- &#103 -carboline alkaloids if accumulated in tissues, as reported elsewhere, might exhibit antioxidant activity.     

Enantioselective Dynamic Process Reduction of α- and β-Tetralone and Stereoinversion of Resulting Alcohols in a Selected Strain Culture

α-Tetralone and β-tetralone were subjected to biotransformation by 14 fungal strains. Enantiomeric purity of the products depended on the reaction time. 3-Day transformation of α-tetralone in Absidia cylindrospora culture gave S-(+)-1,2,3,4-tetrahydro-1-naftol of 92 % ee, whereas longer biotransformation time resulted in decrease of ee value. 3-Day transformation of β-tetralone by the same strain gave predominantly S-(-)-1,2,3,4-tetrahydro-2-naftol, whereas after 9 days of the reaction, the R-enantiomer with 85 % ee was isolated. Transformation of β-tetralone by Chaetomium sp. KCh 6651 gave pure (S)-(-)-1,2,3,4-tetrahydro-2-naftol in high yield at the concentration of 1 g/l. In this process, a non-selective carbonyl reduction was observed, followed by a selective oxidation of the R-alcohol.     

Formaldehyde-derived tetrahydroisoquinolines and tetrahydro-β-carbolines in human urine

Human urine samples were examined for the occurrence of formaldehyde-derived tetrahydroisoquinolines and tetrahydro-β-carbolines generated by condensation of the methanol oxidation product with biogenic amines. Positive results were obtained for the tryptamine condensation product 1,2,3,4-tetrahydro-β-carboline and the serotonine condensation product 6-hydroxy-1,2,3,4-tetrahydro-β-carboline as well as for the condensation products with tyramine, dopamine, adrenaline and noradrenaline 1,2,3,4-tetrahydroisoquinoline, 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, 4,6,7-trihydroxy-1,2,3,4-tetrahydroisoquinoline, and the metabolite 6-methoxy-7-hydroxy-1,2,3,4-tetrahydroisoquinoline. Negative results were obtained for N-methyl-1,2,3,4-tetrahydroisoquinoline and 6,7-di-methoxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-1,2,3,4-tetrahydro-β-carboline, 6-methyl-1,2,3,4-tetrahydro-β-carboline, and 6-methoxy-1,2,3,4-tetrahydro-β-carboline in samples of chronic alcoholics as well as in the urine of healthy volunteers. No correlation between alcohol ingestion or state of alcoholization could be demonstrated.     

Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis

AIM: To identify alkyl hydroperoxide reductase subunit C(AhpC) homologs in Bacillus subtilis(B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria.METHODS: Two AhpC homologs(AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues(C37S, C47 S, C166 S, C37/47 S, C37/166 S, C47/166 S, and C37/47/166 S for AhpC_H1; C52 S, C169 S, and C52/169 S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahp C genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined.RESULTS: Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis. CONCLUSION: AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.     

Design, synthesis, and biological evaluation of novel (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids as selective inhibitors for AKR1B1

New substituted (1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acids were designed as the inhibitor of AKR1B1 based upon the structure of rhetsinine, a minor alkaloidal component of Evodia rutaecarpa, and twenty derivatives were synthesized and evaluated. The most active compound of the series was (2-benzyl-6-methoxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7m), which showed comparable inhibitory activity for AKR1B1 (IC(50)=0.15μM) with clinically used epalrestat (IC(50)=0.1μM). In the view of activity and selectivity, the most potent compound was (2-benzyl-6-carboxy-1-thioxo-1,2,3,4-tetrahydro-β-carbolin-9-yl)acetic acid (7t), which showed strong inhibitory effect (IC(50)=0.17μM) and very high selectivity for AKR1B1 against AKR1A1 (311:1) and AKR1B10 (253:1) compared with epalrestat.     

Urate hydroperoxide oxidizes endothelial cell surface protein disulfide isomerase-A1 and impairs adherence

BackgroundExtracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation.MethodsAmino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively.ResultsUrate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 10³ M⁻¹ s⁻¹. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio.ConclusionsOur results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence.General significanceThese findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.     

Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes. Formation of alkyl and alkoxyl radicals from tert-butyl hydroperoxide and cumene hydroperoxide

The organic hydroperoxides tert-butyl hydroperoxide and cumene hydroperoxide are tumor promoters in the skin of SENCAR mice, and this activity is presumed to be mediated through the activation of the hydroperoxides to free radical species. In this study we have assessed the generation of free radicals from organic hydroperoxides in the target cell (the murine basal keratinocyte) using electron spin resonance. Incubation of primary isolates of keratinocytes from SENCAR mice in the presence of spin traps (5,5-dimethyl-1-pyrroline N-oxide or 2-methyl-2-nitrosopropane) and either tert-butyl hydroperoxide or cumene hydroperoxide resulted in the generation and detection of radical adducts of these spin traps. tert-Butyl alkoxyl and alkyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide were detected shortly after addition of tert-butyl hydroperoxide, whereas only alkyl radical adducts were observed with cumene hydroperoxide. Spin trapping of the alkyl radicals with 2-methyl-2-nitrosopropane led to the identification of methyl and ethyl radical adducts following both tert-butyl hydroperoxide and cumene hydroperoxide exposures. Prior heating of the cells to 100 degrees C for 30 min prevented radical formation. The radical generating capacity of subcellular fractions of these epidermal cells was examined using 5,5-dimethyl-1-pyrroline N-oxide and cumene hydroperoxide, and this activity was confined to the 105,000 X g supernatant fraction.     

Synthesis and in vitro acetylcholinesterase inhibitory activity of some 1-substituted analogues of velnacrine

A number of analogues of 9-amino-1,2,3,4-tetrahydro-1-acridinol (velnacrine), with 1-position substituents other than hydroxy, were prepared and evaluated for in vitro acetylcholinesterase inhibition.     

Distribution of lipoxygenase and hydroperoxide lyase in the leaves of various plant species

The enzyme activity responsible for volatile C₆-aldehyde formation was accompanied by lipoxygenase and hydroperoxide lyase in the green leaves of 28 plant species tested, but the level of each enzyme's activity varied. Lipoxygenase activity rather than hydroperoxide lyase activity appears to affect the overall C₆-aldehyde formation. There was a positive correlation (r = 0.712) between hydroperoxide lyase activity and the chlorophyll content of the green leaves; no correlation was found between lipoxygenase activity and chlorophyll content.     

Design and synthesis of 2,3,4,9-tetrahydro-1H-carbazole and 1,2,3,4-tetrahydro-cyclopenta[b]indole derivatives as non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA polymerase

A novel class of HCV NS5B RNA dependent RNA polymerase inhibitors containing 2,3,4,9-tetrahydro-1H-carbazole and 1,2,3,4-tetrahydro-cyclopenta[b]indole scaffolds were designed and synthesized. Optimization of the aromatic region showed preference for 5,8-disubstitution pattern in both the scaffolds examined while favoring the n-propyl moiety for the C-1 position. 1,2,3,4-tetrahydro-cyclopenta[b]indole scaffold was slightly more potent than the corresponding 2,3,4,9-tetrahydro-1H-carbazole and analogue 36 displayed an IC50 of 550 nM against HCV NS5B enzyme.     

Lipid hydroperoxide levels in plant tissues

Hydroperoxides are the primary oxygenated products of polyunsaturated fatty acids and are key intermediates in the octadecanoid signalling pathway in plants. Lipid hydroperoxides (LHPO) were determined spectrophotometrically based on their reaction with an excess of Fe(2+)at low pH in the presence of the dye xylenol orange. Triphenylphosphine-mediated hydroxide formation was used to authenticate the signal generated by the hydroperoxides. The method readily detected lipid peroxidation in Phaseolus: microsomes, senescing potato leaves and in a range of other plant tissues including Phaseolus hypocotyls (26+/-5 nmol g(-1) FW), Alstroemeria floral tissues (sepals 66+/-13 nmol g(-1) FW petals 49+/-6 nmol g(-1) FW), potato leaves (334+/-75 nmol g(-1) FW), broccoli florets (568+/-68 nmol g(-1) FW) and Chlamydomonas cells (602+/-40 nmol g(-1) FW). Relative to the total fatty acid content of the tissues, the % LHPO was within the range of 0.6-1.7% for all tissue types (photosynthetic and non-photosynthetic) and represents the basal oxidation level of membrane fatty acids in plant cells. In order to relate the levels of LHPO to specific signalling pathways, transgenic potato plant lines were used in which lipoxygenase (LOX) (responsible for hydroperoxide biosynthesis) and hydroperoxide lyase (a route of hydroperoxide degradation) activities were largely reduced by an antisense-mediated approach. While the LHPO levels were similar to wild type in the individual LOX antisensed plants, basal LHPO levels, by contrast, were elevated by 38% in transgenic potato leaves antisensed in hydroperoxide lyase, indicating a role for this enzyme in the maintenance of cellular levels of LHPOs.     

Structural snapshots of yeast alkyl hydroperoxide reductase Ahp1 peroxiredoxin reveal a novel two-cysteine mechanism of electron transfer to eliminate reactive oxygen species

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that protect cells against reactive oxygen species and are involved in cellular signaling pathways. Alkyl hydroperoxide reductase Ahp1 belongs to the Prx5 subfamily and is a two-cysteine (2-Cys) Prx that forms an intermolecular disulfide bond. Enzymatic assays and bioinformatics enabled us to re-assign the peroxidatic cysteine (C_P) to Cys-62 and the resolving cysteine (C_R) to Cys-31 but not the previously reported Cys-120. Thus Ahp1 represents the first 2-Cys Prx with a peroxidatic cysteine after the resolving cysteine in the primary sequence. We also found the positive cooperativity of the substrate t-butyl hydroperoxide binding to Ahp1 homodimer at a Hill coefficient of ∼2, which enabled Ahp1 to eliminate hydroperoxide at much higher efficiency. To gain the structural insights into the catalytic cycle of Ahp1, we determined the crystal structures of Ahp1 in the oxidized, reduced, and Trx2-complexed forms at 2.40, 2.91, and 2.10 Å resolution, respectively. Structural superposition of the oxidized to the reduced form revealed significant conformational changes at the segments containing C_P and C_R. An intermolecular C_P-C_R disulfide bond crossing the A-type dimer interface distinguishes Ahp1 from other typical 2-Cys Prxs. The structure of the Ahp1-Trx2 complex showed for the first time how the electron transfers from thioredoxin to a peroxidase with a thioredoxin-like fold. In addition, site-directed mutagenesis in combination with enzymatic assays suggested that the peroxidase activity of Ahp1 would be altered upon the urmylation (covalently conjugated to ubiquitin-related modifier Urm1) of Lys-32.     

Synthesis and evaluation of 1,2,3,4-tetrahydro-1-acridone analogues as potential dual inhibitors for amyloid-beta and tau aggregation

Amyloid-β (Aβ) and tau protein are two crucial hallmarks in Alzheimer’s disease (AD). Their aggregation forms are thought to be toxic to the neurons in the brain. A series of new 1,2,3,4-tetrahydro-1-acridone analogues were designed, synthesized, and evaluated as potential dual inhibitors for Aβ and tau aggregation. In vitro studies showed that compounds 25–30 (20?μM) with N-methylation of the quinolone ring effectively inhibited Aβ_1-42 aggregation by 84.7%–99.5% and tau aggregation by 71.2%–101.8%. Their structure-activity relationships are discussed. In particular, 30 could permeate the blood-brain barrier, bind to Aβ_1-42 and tau, inhibit Aβ_1-42 β-sheets formation, and prevent tau aggregation in living cells.     

SAR studies of capsazepinoid bronchodilators. Part 1: The importance of the catechol moiety and aspects of the B-ring structure

Capsazepine as well as its derivatives and analogues are general inhibitors of constriction of human small airways. From a systematic variation of the capsazepine structure, divided into four regions, SARs were established. This part concerns the catechol moiety of the A-ring as well as the 2,3,4,5-tetrahydro-1H-2-azepine moiety (the B-ring) of capsazepine. It is revealed that a conformational constrain (as a fused ring) is important and that compounds with a six-membered B-ring (as a 1,2,3,4-tetrahydroisoquinoline) in general are more potent than the corresponding isoindoline, 2,3,4,5-tetrahydro-1H-2-benzazepine and 2,3,4,5-tetrahydro-1H-3-benzazepine derivatives.     

3-Oxo-2-piperazinyl acetamides as potent bradykinin B1 receptor antagonists for the treatment of pain and inflammation

The discovery of novel and highly potent oxopiperazine based B1 receptor antagonists is described. Compared to the previously described arylsulfonylated (R)-3-amino-3-phenylpropionic acid series, the current compounds showed improved in vitro potency and metabolic stability. Compound 17, 2-((2R)-1-((4-methylphenyl)sulfonyl)-3-oxo-2-piperazinyl)-N-((1R)-6-(1-piperidinylmethyl)-1,2,3,4-tetrahydro-1-naphthalenyl)acetamide, showed EC₅₀ of 10.3 nM in a rabbit biochemical challenge model. The practical syntheses of chiral arylsulfonylated oxopiperazine acetic acids are also described.