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Mechanism of the reaction of ebselen with endogenous thiols: Dihydrolipoate is a better cofactor than glutathione in the peroxidase activity of ebselen
Abstract
The therapeutic effect of ebselen has been linked to its peroxidase activity. In the present study, the peroxidase activity of ebselen toward H2O2 with the endogenous thiols GSH and dihydrolipoate [L(SH)2] as cofactors was determined. When GSH was used, peroxide removal was described by a ter uni ping pong mechanism with Dalziel coefficients for GSH and H2O2 of 0.165 +/- 0.011 and 0.081 +/- 0.005 mM min, respectively. When L(SH)2 was used, peroxidase activity was independent of the concentration of L(SH)2 in the concentration range studied (5 microM to 2 mM) and peroxide removal was only dependent on the concentration of H2O2 and ebselen, with the second-order rate constant being 12.3 +/- 0.8 mM-1 min-1. To elucidate the difference between GSH and L(SH)2, the molecular mechanism of the peroxidase activity of ebselen was investigated, using UV spectrophotometry, high pressure liquid chromatography, 77Se NMR, and mass spectrometry. GSH was found to react quickly with ebselen to give a selenenyl sulfide, an adduct of GSH to ebselen. Subsequently, the GSH-selenenyl sulfide is converted into the diselenide of ebselen. Finally the diselenide reacts with a peroxide and ebselen is regenerated. The formation by GSH of the diselenide from the GSH-selenenyl sulfide of ebselen is slow and linearly dependent on the concentration of free thiol; however, no net consumption of GSH was observed. Furthermore, it is likely that a selenol is an intermediate in diselenide formation. After reaction between ebselen and L(SH)2 the diselenide of ebselen was immediately detected. The fast formation of the diselenide with L(SH)2 versus the slow formation of the diselenide with GSH accounts for our observation that L(SH)2 is a better cofactor than GSH in the peroxidase activity of ebselen. Our results suggest that the interaction between ebselen and L(SH)2 might be of major importance in the mechanism by which ebselen exerts its therapeutic effect.
... As a direct method, the degradation of H 2 O 2 can be determined by following its concentration versus time, according to the Hildebrandt and Roots method, based on the H 2 O 2 -mediated oxidation of Fe 2+ to Fe 3+ in turn complexed to thiocyanate and quantified spectrophotometrically at 480 nm [71]. This method was used for the evaluation of ebselen using as thiol probe, besides GSH, lipoic acid [72]. NMRbased assays were developed starting from the seminal work of Engman, who moni- tored the conversion of thiol to disulfide by the integration of key peaks in the 1D 1 H NMR; the integral area was then plotted against time to give a linear correlation. ...
... The most important aspect in this approach is the choice of the thiol probe, which needs to be selected among those whose spontaneous oxidation to disulfide is slow in the presence of oxidants but in absence of the catalyst. As example, GSH is not exploitable since its oxidation in water containing H 2 O 2 at pH = 7 is complete in 15 min [72]. The thiol probes that have been used in the NMR-based assays are summarized in Table 2.2. ...
... Besides the GPx-mimic activity, ebselen behaves as an antioxidant because it protected rat liver microsomes [36] and isolated hepatocytes [80] against ascorbate/Fe-induced lipid peroxidation. Besides GSH, other thiols were also employed to assist the ebselen-mediate reduction of hydrogen peroxide, such as N-acetyl cysteine (IV) [81], dithiothreitol (VIII) [80], and dihydrolipoate [72]. ...
After 1984, when ebselen was tested as a mimetic of the key antioxidant enzyme glutathione peroxidase (GPx), a plethora of organoselenium compounds have been synthesized and tested for various pharmacological purposes. Here a brief overview of the most important achievements in bioactive organoselenium small molecules is given, with particular emphasis on the GPx-like as well as to the antiviral, antibacterial, antifungal, and antiprotozoal activities. While historical information is given to help contextualize the content, the most recent literature is comprehensively discussed.
... 14) formed heavier complexes under non-denaturing conditions, but not under denaturing conditions (Fig. 1h). Ebselen contains selenium (Fig. 1a), which can form a selenylsulfide (-Se-S-) bond 18,19 . For bovine IMPase, alkylation of cysteine 218 with the non-selective agent N-ethylmaleimide inhibited activity 20 . ...
... These data demonstrate that inhibition of IMPase by ebselen requires not just the presence of an electrophilic selenium atom but also an appropriate chemical scaffold. Unlike the case for most other proteins when covalently linked to ebselen 18,19,22 , inactivation of IMPase was not reversed by post-incubation with the sulfhydryl-reducing agents glutathione (GSH) and dithiothreitol (DTT) (Fig. 1m). Pre-incubation of ebselen with the reducing agents did, however, reduce inhibition ( Fig. 1n) as described in detail below. ...
... That IMPase inhibition by ebselen was detected in the ex vivo experiments (Fig. 2d,e) is revealing in regard to the likely chemical form of ebselen in intact cells in vivo, as its selenium atom can exist in higher or lower oxidation states, and these have ARTICLE different reactivities 18,19 . Incubation of ebselen with reduced GSH forms ebselen-GSH selenenylsulphide, whereas incubation with DTT reduces ebselen to its selenol and diselenide (Fig. 1a,l) 18,19 . ...
Vincent Van Gogh painted for just 10 years, but produced over 2000 works of art in periods of intense productivity and creativity. However, during these creative periods, his personal life was one of chaos with poor and impulsive decisions relating to finance, career, business, substance abuse, sexuality and romance (sending his severed ear to the object of his affection), and between these periods he was institutionalized with crushing depressions and he eventually took his own life. It is likely that he suffered from what we now know as bipolar disorder. Insight into mood is not easily conveyed, but we can get a sense of Van Gogh's mood extremes from two of his paintings, both with the theme of a starry night, but one suggesting exuberant mania and one gloomy depression. Surprisingly, the best treatment for bipolar disorder still remains the one first discovered over 60 years ago: the cation lithium. Nevertheless, lithium's side effects and toxicity have spurred the search for a replacement.
... Ebselen contains selenium (Fig. 1a), which can form a selenylsulfide (-Se-S-) bond 18,19 . For bovine IMPase, alkylation of cysteine 218 with the non-selective agent Nethylmaleimide inhibited activity 20 . ...
... These data demonstrate that inhibition of IMPase by ebselen requires not just the presence of an electrophilic selenium atom but also an appropriate chemical scaffold. Unlike the case for most other proteins when covalently linked to ebselen 18,19,22 , inactivation of IMPase was not reversed by post-incubation with the sulfhydryl reducing agents glutathione and dithiothreitol (Fig. 1m). Pre-incubation of ebselen with the reducing agents did, however, reduce inhibition (Fig. 1n) as described in detail below. ...
... That IMPase inhibition by ebselen was detected in the ex vivo experiments (Fig. 2d,e) is revealing in regard to the likely chemical form of ebselen in intact cells in vivo, as its selenium atom can exist in higher or lower oxidation states, and these have different reactivities 18,19 . Incubation of ebselen with reduced glutathione forms ebselen-glutathione selenenylsulphide, whereas incubation with dithiothreitol reduces ebselen to its selenol and diselenide (Fig. 1a,l) 18,19 . ...
Lithium is the most effective mood stabilizer for the treatment of bipolar disorder, but it is toxic at only twice the therapeutic dosage and has many undesirable side effects. It is likely that a small molecule could be found with lithium-like efficacy but without toxicity through target-based drug discovery; however, therapeutic target of lithium remains equivocal. Inositol monophosphatase is a possible target but no bioavailable inhibitors exist. Here we report that the antioxidant ebselen inhibits inositol monophosphatase and induces lithium-like effects on mouse behaviour, which are reversed with inositol, consistent with a mechanism involving inhibition of inositol recycling. Ebselen is part of the National Institutes of Health Clinical Collection, a chemical library of bioavailable drugs considered clinically safe but without proven use. Therefore, ebselen represents a lithium mimetic with the potential both to validate inositol monophosphatase inhibition as a treatment for bipolar disorder and to serve as a treatment itself.
... Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is an organoselenium compound that is known to possess anti-atherosclerotic, anti-inflammatory, antioxidative, cytoprotective, antimutagenic and anti-lipoperoxidative properties [1][2][3][4]. Several studies have demonstrated that ebselen, due to its highly electrophilic nature, interacts with cysteine rich proteins (such as thioredoxin) and non-proteins (thiols) [2,[5][6][7][8][9][10][11]. Ebselen specifically interacts with free thiols such as glutathione (GSH) to form ebselen selenenyl sulfide; this intermediate catalyzes reactive ...
... Interestingly, ebselen selenenyl sulfide can be reduced by GSH to form ebselen selenol. This particular intermediate functions as a ROS scavenger, and thereby protects the cell from free radical damage [2,6,7]. As a clinically safe molecule, ebselen has been investigated for the treatment of various ailments such as arthritis, stroke, cardiovascular disease and cancer [2,[12][13][14][15]. ...
Background:
Ebselen, an organoselenium compound and a clinically safe molecule has been reported to possess potent antifungal activity, but its antifungal mechanism of action and in vivo antifungal activity remains unclear.
Methods:
The antifungal effect of ebselen was tested against Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococcus neoformans, and C. gattii clinical isolates. Chemogenomic profiling and biochemical assays were employed to identify the antifungal target of ebselen. Ebselen's antifungal activity in vivo was investigated in a Caenorhabditis elegans animal model.
Results:
Ebselen exhibits potent antifungal activity against both Candida spp. and Cryptococcus spp., at concentrations ranging from 0.5-2μg/ml. Ebselen rapidly eradicates a high fungal inoculum within two hours of treatment. Investigation of the drug's antifungal mechanism of action indicates that ebselen depletes intracellular glutathione (GSH) levels, leading to increased production of reactive oxygen species (ROS), and thereby disturbs the redox homeostasis in fungal cells. Examination of ebselen's in vivo antifungal activity in two Caenorhabditis elegans models of infection demonstrate that ebselen is superior to conventional antifungal drugs (fluconazole, flucytosine and amphotericin) in reducing Candida and Cryptococcus fungal load.
Conclusion:
Ebselen possesses potent antifungal activity against clinically relevant isolates of both Candida and Cryptococcus by regulating GSH and ROS production. The potent in vivo antifungal activity of ebselen supports further investigation for repurposing it for use as an antifungal agent.
General significance:
The present study shows that ebselen targets glutathione and also support that glutathione as a potential target for antifungal drug development.
... A clinical trial of ebselen as a therapy for SARS-CoV-2 was also announced recently [60]. Scheme 1 shows some of the key pathways that have been proposed in its catalytic antioxidant mechanism [61][62][63][64][65][66][67][68][69][70]. Since various groups have employed different thiols, peroxides, conditions and assay techniques in their mechanistic investigations, the results have not always been consistent. ...
The antioxidant drug ebselen has been widely studied in both laboratories and in clinical trials. The catalytic mechanism by which it destroys hydrogen peroxide via reduction with glutathione or other thiols is complex and has been the subject of considerable debate. During reinvestigations of several key steps, we found that the seleninamide that comprises the first oxidation product of ebselen underwent facile reversible methanolysis to an unstable seleninate ester and two dimeric products. In its reaction with benzyl alcohol, the seleninamide produced a benzyl ester that reacted readily by selenoxide elimination, with formation of benzaldehyde. Oxidation of ebselen seleninic acid did not afford a selenonium seleninate salt as previously observed with benzene seleninic acid, but instead generated a mixture of the seleninic and selenonic acids. Thiolysis of ebselen with benzyl thiol was faster than oxidation by ca. an order of magnitude and produced a stable selenenyl sulfide. When glutathione was employed, the product rapidly disproportionated to glutathione disulfide and ebselen diselenide. Oxidation of the S-benzyl selenenyl sulfide, or thiolysis of the seleninamide with benzyl thiol, afforded a transient thiolseleninate that also readily underwent selenoxide elimination. The S-benzyl derivative disproportionated readily when catalyzed by the simultaneous presence of both the thiol and triethylamine. The phenylthio analogue disproportionated when exposed to ambient or UV (360 nm) light by a proposed radical mechanism. These observations provide additional insight into several reactions and intermediates related to ebselen.
... Additionally, we identified silver sulfadiazine and ebselen as compounds with the surprising capacity to nearly completely suppress GTP uptake, which was apparently ''rescued'' by RGS4 ( Figure S6A). However, since both of these compounds are known to be covalent modifiers of cysteine groups, 33 we next tested the general reducing agents DTT and b-mercaptoethanol, finding that they produced a similar ''rescue'' ( Figure S6B). We thus must conclude that the RGS4 ''rescue'' action in this case is simply mediated through providing competing cysteine residues, thus protecting Gɑo from an excess of silver sulfadiazine or ebselen, and a similar effect can likely be achieved by any protein with cysteines exposed. ...
Background:
The GNAO1 gene, encoding the major neuronal G protein Gαo, is mutated in a subset of pediatric encephalopathies. Most such mutations consist of missense variants.
Methods:
In this study, we present a precision medicine workflow combining next-generation sequencing (NGS) diagnostics, molecular etiology analysis, and personalized drug discovery.
Findings:
We describe a patient carrying a de novo intronic mutation (NM_020988.3:c.724-8G>A), leading to epilepsy-negative encephalopathy with motor dysfunction from the second decade. Our data show that this mutation creates a novel splice acceptor site that in turn causes an in-frame insertion of two amino acid residues, Pro-Gln, within the regulatory switch III region of Gαo. This insertion misconfigures the switch III loop and creates novel interactions with the catalytic switch II region, resulting in increased GTP uptake, defective GTP hydrolysis, and aberrant interactions with effector proteins. In contrast, intracellular localization, Gβγ interactions, and G protein-coupled receptor (GPCR) coupling of the Gαo[insPQ] mutant protein remain unchanged.
Conclusions:
This in-depth analysis characterizes the heterozygous c.724-8G>A mutation as partially dominant negative, providing clues to the molecular etiology of this specific pathology. Further, this analysis allows us to establish and validate a high-throughput screening platform aiming at identifying molecules that could correct the aberrant biochemical functions of the mutant Gαo.
Funding:
This work was supported by the Joint Seed Money Funding scheme between the University of Geneva and the Hebrew University of Jerusalem.
... It was reported by Cotgreave et al. [263], Maiorino et al. [264], and Morgenstern et al. [265] that selenol is the prime molecular class that is accountable for helping ebselen in mimicking the activity of GPx. GSH replacement with dihydrolipoate (134) expands the activity of ebselen in peroxidase (Scheme 31) [266]. ...
Selenium (Se) is known for its beneficial biological roles for several years but interest in this trace element has seen significant increase in the past couple of decades. It has been reported to be a part of important bioactive organic compounds, such as selenoproteins and amino acids including selenocysteine (SeCys), selenomethionine (SeMet), selenazolidine (SeAzo), and selenoneine. The traditional Se supplementations (primarily as selenite, and selenomethionine), though have been shown to carry some benefits, also have associated toxicities thereby paving the way for the organoselenium compounds, especially the selenoproteins and peptides (SePs/SePPs) that offer several health benefits beyond fulfilling the elementary nutritional Se needs. This review aims to showcase the applications of selenium-containing peptides that have been reported in recent decades. Herein this article summarize their bioactivities including neuroprotective, antiinflammatory, anticancer, antioxidant, hepatoprotective, and immunomodulatory roles. This will offer the readers a sneak peek into the current advancements to invoke further developments in this emerging research area.
... The catalytic dyad (Cys145 and His41) was considered neutral, as highlighted in previous studies [31,32]. An adduct between EbSe and cysteine (EbSe-Cys) was used in the docking simulations to mimic the putative metabolites of EbSe [33][34][35]. The tridimensional model of the ligands (EbSe and EbSe-Cys) were created with Avogadro and MOPAC (PM6 method) [36,37]. ...
The main protease (Mpro) of SARS-CoV-2 is a current target for the inhibition of viral replication. Through a combined Docking and Density Functional Theory (DFT) approach, we investigated in-silico the molecular mechanism by which ebselen (IUPAC: 2-phenyl-1,2-benzoselenazol-3-one), the most famous and pharmacologically active organoselenide, inhibits Mpro. For the first time, we report on a mechanistic investigation in an enzyme for the formation of the covalent -S-Se- bond between ebselen and a key enzymatic cysteine. The results highlight the strengths and weaknesses of ebselen and provide hints for a rational drug design of bioorganic selenium-based inhibitors.
... Hbonds, electrostatic (charge-charge) and hydrophobic (π-π) interactions, besides the zinc coordination, are represented by green, orange, purple, and blue dotted lines, respectively; all distances are in Å. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) atom of the Se-S bond, leading to the disulfide bridge (S-S) formation, i.e., thiol oxidation, and the release of zinc ion [7,99,108,[111][112][113]. In fact, the distances between the S atoms are 3.7-4.6 ...
Organoselenium compounds present many pharmacological properties and are promising drugs. However, toxicological effects associated with inhibition of thiol-containing enzymes, such as the δ-aminolevulinic acid dehydratase (δ-AlaD), have been described. The molecular mechanism(s) by which they inhibit thiol-containing enzymes at the atomic level, is still not well known. The use of computational methods to understand the physical-chemical properties and biological activity of chemicals is essential to the rational design of new drugs. In this work, we propose an in silico study to understand the δ-AlaD inhibition mechanism by diphenyl diselenide (DPDS) and its putative metabolite, phenylseleninic acid (PSA), using δ-AlaD enzymes from Homo sapiens (Hsδ-AlaD), Drosophila melanogaster (Dmδ-AlaD) and Cucumis sativus (Csδ-AlaD). Protein modeling homology, molecular docking, and DFT calculations are combined in this study. According to the molecular docking, DPDS and PSA might bind in the Hsδ-AlaD and Dmδ-AlaD active sites interacting with the cysteine residues by Se…S interactions. On the other hand, the DPDS does not access the active site of the Csδ-AlaD (a non-thiol protein), while the PSA interacts with the amino acids residues from the active site, such as the Lys291. These interactions might lead to the formation of a covalent bond, and consequently, to the enzyme inhibition. In fact, DFT calculations (mPW1PW91/def2TZVP) demonstrated that the selenylamide bond formation is energetically favored. The in silico data showed here are in accordance with previous experimental studies, and help us to understand the reactivity and biological activity of organoselenium compounds.
... The oxidation of a diselenide is also postulated in the catalytic cycle of ebselen 8 [18,155,156,187]. ...
Organodiselenides are an important class of compounds characterized by the presence of two adjacent covalently bonded selenium nuclei. Among them, diaryldiselenides and their parent compound diphenyl diselenide, attract continuing interest in chemistry as well as in close disciplines like medicinal chemistry, pharmacology and biochemistry. A search in SCOPUS database has revealed that in the last three years 105 papers have been published on the archetypal diphenyl diselenide and its use in organic catalysis and drug tests. The reactivity of the Se-Se bond and the redox properties of selenium make diselenides efficient catalysts for numerous organic reactions, such as Bayer-Villiger oxidations of aldehydes/ketones, epoxidations of alkenes, oxidations of alcohols and nitrogen containing compounds. In addition, organodiselenides might find application as mimics of glutathione peroxidase (GPx), a family of enzymes, which, besides performing other functions, regulate the peroxide tone in the cells and control the oxidative stress level. In this review, the essential synthetic and reactivity aspects of organoselenides are collected and rationalized using the results of accurate computational studies, which have been carried out mainly in the last two decades. The results obtained in silico provide a clear explanation of the anti-oxidant activity of organodiselenides and more in general of their ability to reduce hydroperoxides. At the same time, they are useful to gain insight into some aspects of the enzymatic activity of the GPx, inspiring novel elements for rational catalyst and drug design.
... The direct reaction of the same compound with H 2 O 2 (Scheme 1) was also studied in silico, 28 but was found to be disfavored: it resulted in the formation of a selenolseleninate with a strongly distorted structure, leading the authors to exclude the feasibility of this pathway. Moreover, there is still a debate on the product of this direct oxidation by H 2 O 2 : experimental studies on ebselen ‡ [29][30][31] suggest that the reaction of its diselenide intermediate, obtained by dimerization, with H 2 O 2 leads to the formation of a selenenic anhydride, 32,33 although no evidence is provided. Conversely, computational studies 34,35 propose the initial formation of a selenolseleninate. ...
The reactivity of diselenides and ditellurides of general formula (RX)2 (X=Se, Te; R=H, CH3, C6H5) toward hydrogen peroxide was studied through a computational approach based on accurate Density Functional Theory (DFT) calculations. The aliphatic and aromatic dichalcogenides have been chosen in light of their activity in glutathione peroxidase (GPx)-like catalytic cycles and their promising features as efficient antioxidant compounds. The reaction products, the energetics and the mechanistic details of these oxidations are discussed. Analogous disulfides are included in our analysis for completeness. We find that the barrier for oxidation of dichalcogenides decreases from disufides to diselenides to ditellurides. On the other hand, variation of the substituents at the chalcogen nucleus has relatively little effect on the reactivity.
... Good antioxidants form vitamin E and vitamin C analogues [53,54]. Ebselen is a newly designed anti-inflammatory agent that has peroxidase activity [55]. Also, SOD enzymatic activity has been mimicked [56] as, for example, with Cu(II)2(3,5-DIPSh. ...
... Some experimental studies [35][36][37][38][39] have suggested the appearance of diselenides during the catalytic cycle, which Pearson and Boyd therefore also included in their studies [33,34]. Interestingly, the study on the small model systems indicates that dimethyl diselenide would be rather active in reducing hydrogen peroxide, having a barrier of 35.3 kcal mol -1 that is further lowered to 29.6 kcal mol -1 when solvent effects are included. ...
Organoselenium compounds can reduce H2O2 and organic hydroperoxides mimicking the antioxidant activity of glutathione peroxidase (GPx). This catalytic process reduces the peroxides, which play a key role in oxidative stress, thus inhibiting their harmful action. Thus, it is not surprising that a lot of effort has been devoted toward the synthesis and design of numerous GPx mimics, which, nevertheless, show minor efficiency when compared to the native enzyme. In this context, quantum chemistry tools are an important support to rational drug design, because they allow to investigate in detail the structural and electronic properties of the organochalcogen compound with the aim of establishing links with its potential catalytic activity. In this review, we have collected the information from the available quantum chemistry studies about the reactivity of organic selenides with peroxides and thiols, delineating the analysis on monoselenides and ebselen, which will be critically discussed and gathered for a more complete overview of their GPx-like activity; few novel results will be presented to interpret very recent experimental mechanistic findings.
... Indeed, the activity of untreated enzyme could be partly restored by incubation with 1 mm dithiothreitol (35±45% of the not PN-treated control). Also 10 mm ebselen, which is known to form adducts with thiols [55,56] and to react with an essential thiol group of the NADPH reductase [54], was found to block the enzyme activity completely (not shown). Based on these observations a thioldependent inactivation is likely, especially as thiol oxidation is a common modification mediated by PN [10±12]. ...
Peroxynitrite (PN) is likely to be generated in vivo from nitric oxide and superoxide. We have previously shown that prostacyclin synthase, a heme-thiolate enzyme essential for regulation of vascular tone, is nitrated and inactivated by submicromolar concentrations of PN [Zou, M.-H. & Ullrich, V. (1996) FEBS Lett.382, 101–104] and we have studied the effect of heme proteins on the PN-mediated nitration of phenolic compounds in model systems [Mehl, M., Daiber, A. & Ullrich, V. (1999) Nitric Oxide: Biol. Chem.2, 259–269]. In the present work we show that bolus additions of PN or PN-generating systems, such as SIN-1, can induce the nitration of P450BM-3 (wild-type and F87Y variant), for which we suggest an autocatalytic mechanism. HPLC and MS-analysis revealed that the wild-type protein is selectively nitrated at Y334, which was found at the entrance of a water channel connected to the active site iron center. In the F87Y variant, Y87, which is directly located at the active site, was nitrated in addition to Y334. According to Western blots stained with a nitrotyrosine antibody, this nitration started at 0.5 µm of PN and was half-maximal between 100 and 150 µm of PN. Furthermore, PN caused inactivation of the P450BM-3 monooxygenase as well as the reductase activity with an IC50 value of 2–3 µm. As two thiol residues/protein molecule were oxidized by PN and the inactivation was prevented by GSH or dithiothreitol, but not by uric acid (a powerful inhibitor of the nitration), our data strongly indicate that the inactivation is due to thiol oxidation at the reductase domain rather then to nitration of Y residues. Stopped-flow data presented here support our previous hypothesis that ferryl-species are involved as intermediates during the reactions of P450 enzymes with PN.
... Eb has previously been shown to inhibit protein activity by the formation of a covalent bond between its selenium and the thiols in cysteine residues. 28,30,36,37 Alternatively, Eb can also alter protein activity independently of selenium through the remainder of its chemical scaffold. 30,38 To assess the importance of the oxidation state of Eb for its ability to inhibit cdiGMP-receptor interactions, we determined the ability of ebselen oxide (EbO) to inhibit cdiGMP binding to the same panel of proteins described above ( Figure 4A, B). ...
The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.
... In earlier work on the mechanism of the GPxlike activity of ebselen, Fischer and Dereu proposed, on the basis of their 77 Se NMR study [36], the functioning of two catalytic cycles ( Fig. 1, Cycles A and B) dependent on whether the hydroperoxide (Fig. 1, Cycle A) or the thiol (Fig. 1, Cycle B) occurs in excess over the other reaction partner. On the other hand, later work of other groups has unequivocally established the transient formation of the selenol in aqueous systems containing glutathione [37,38]. In this way, Cycle C would be operative under the premise that both ebselen selenol and ebselen diselenide are required intermediates. ...
Selenium represents an essential element for organisms as various diseases can result from selenium deficiency. As a consequence, selenium-containing heterocycles are of considerable biochemical and pharmacological relevance. Selenium-containing heterocycles are often less stable than the corresponding sulfur analogues. Therefore, the investigation of new methods for the synthesis of small selenium-containing building blocks is of considerable interest. This review describes the use of biologically significant selenium-containing heterocycles from the viewpoint of chemical structures.
... The peroxidase activity proceeds by a ter uni ping-pong mechanism and the kinetics can be described using the Dalziel equation. In the peroxidase activity of ebselen, there is a substantial difference in the Dalziel constants of thiols [14]. Because the rate constants are determined by directly monitoring hydrogen peroxide, the problems addressed in this paper do not apply. ...
During many processes in the human body, reactive species are formed. Antioxidants, produces by the human body or available through the diet, can protect human cells against the damage caused by these reactive species. The quest for the most efficient antioxidant is ongoing as countless compounds are screened for their antioxidant activity in vitro by competition assays. Unfortunately, the results of the different competition assays are not in consensus for the ranking of antioxidants according to their activity. We found that the antioxidant activity, described by its EC50, is dependent on
• the reaction rate constant of the detector,
the reaction rate constant of the antioxidant under test,
• the concentration of reactive species used,
• the stoichiometry of the reaction of the antioxidant with the reactive species, and
• the total capacity of the antioxidant.
We also found that there are two kinds of antioxidant activity assays: the competition assay and the capacity assay. In the competition assay, the reaction rate constant of the antioxidant is the discriminating factor for the antioxidant activity. In the capacity assay, the capacity of the antioxidant determines the antioxidant activity. A simple test system is developed to indicate the discriminating factor in the assay used. To give an adequate description of the antioxidant activity, detailed knowledge of the in vivo situation and a careful design of the in vitro assay are necessary.
... To evaluate the peroxynitrite scavenging activity of wine, the activity of wine is compared to that of ebselen. Ebselen is a selenium-containing compound that displays a glutathione peroxidase activity (Haenen et al., 1990). Recently, it has been reported to be an excellent peroxynitrite scavenger (Masumoto, 1996). ...
Keywords: Antioxidant; flavonoid; polyphenol; peroxynitrite; wine
... This rapid conversion of the DTTquercetin adduct explains why, in contrast to DTT-monoHER, this adduct was not found when quercetin was oxidized in the presence of DTT. This reaction is comparable to the reactions of DTT with disulfides and other compounds that also proceed relatively fast, with a DTT adduct as an intermediate [30]. ...
During the scavenging of free radicals flavonoids are oxidized to electrophilic quinones. Glutathione (GSH) can trap these quinones, thereby forming GSH-flavonoid adducts. The aim of this study was to compare the stability of the GSH-flavonoid adduct of 7-mono-O-(β-hydroxyethyl)rutoside (monoHER) with that of quercetin. It was found that GSH-quercetin reacts with the thiol N-acetyl-L-cysteine (NAC) to form NAC-quercetin, whereas GSH-monoHER does not react with NAC. In addition, the adduct of the monoHER quinone with the dithiol dithiothreitol (DTT) is relatively stable, whereas the DTT-quercetin adduct is readily converted into quercetin and DTT disulfide. These differences in reactivity of the thiol-flavonoid adducts demonstrate that GSH-monoHER is much more stable than GSH-quercetin. This difference in reactivity was corroborated by molecular quantum chemical calculations. Thus, although both flavonoid quinones are rapidly scavenged by GSH, the advantage of monoHER is that it forms a stable conjugate with GSH, thereby preventing a possible spread of toxicity. These findings demonstrate that even structurally comparable flavonoids behave differently, which will be reflected in the biological effects of these flavonoids.
... Due to the introduction of ebselen segments, the spectra of e-PEI solutions exhibit two bands. While the absorption at 280 nm is from the benzene ring, the absorption band at 350 nm is from the isoselenazol ring of the ebselen segments [27]. The ratio of C-ebselen moieties in the polymers calculated from the absorption differences at 280 nm was 2.2, which is consistent with the ICP results. ...
A carboxyl-ebselen-based layer-by-layer (LbL) film was fabricated by alternatively assembling carboxyl-ebselen immobilized polyethylenimine (e-PEI) and alginate (Alg) onto substrates followed by salt annealing and cross-linking. The annealed films exhibiting significantly improved stability are capable of generating nitric oxide (NO) from endogeneous S-nitrosothiols (RSNOs) in the presence of a reducing agent. The NO generation behaviors of different organoselenium species in solution phase are compared and the annealing mechanism to create stable LbL films is studied in detail. An LbL film coated polyurethane catheter is capable of generating physiological levels of NO from RSNOs even after blood soaking for 24 h, indicating potential antithrombotic applications of the coating. Further, the LbL film is also demonstrated to be capable of reducing living bacterial surface attachment and killing a broad spectrum of bacteria, likely through generation of superoxide (O(2)(·-)) from oxygen. This type of film is expected to have potential application as an antithrombotic and antimicrobial coating for different biomedical device surfaces.
... It is important to point out that contrasting to the reaction catalyzed by the enzyme, which contains binding sites conferring substrate speciWcity, ebselen and other organoselenium compounds can utilize a variety of thiols (Wendel et al. 1984;Müller et al. 1984;Fischer and Dereu 1987;Engman et al. 1992;Iwaoka and Tomoda 1994;Mugesh et al. 2001), in addition to GSH, as a substrate (Cotgreave et al. 1987;Maiorino et al. 1988;Haenen et al. 1990). ...
The advance in the area of synthesis and reactivity of organoselenium, as well as the discovery that selenium was the cause of severe intoxication episodes of livestock in the 1930s and the subsequent determination that selenium was an essential trace element in the diet for mammals, has motivated intense studies of the biological properties of both organic and inorganic selenium compounds. In this review, we shall cover a wide range of toxicological and pharmacological effects, in which organoselenium compounds are involved but the effects of inorganic compounds were not discussed in detail here. The molecular toxicity of inorganic selenium was described in relation to its interaction with endogenous -SH groups to allow a comparison with that of synthetic organoselenium compounds. Furthermore, in view of the recent points of epidemiological evidence that overexposure to selenium can facilitate the appearance of chronic degenerative diseases, we also briefly revised the history of selenium toxicity and physiology and how environmental selenium can reach inside the mammalian cells. The biological narrative of the element selenium, in the last century, has been marked by a contrast between its toxic and its beneficial effects. Thus, the potential therapeutic use of simple organoselenium compounds has not yet been sufficiently explored and, consequently, we cannot discard this class of compounds as promising pharmaceutical agents. In effect, the future of the organochalcogens as pharmacological agents will depend on more detailed toxicological studies in the oncoming years.
Organoselenium compounds are well known for their effective oxygen-transfer abilities in both stoichiometric and catalytic amounts. The number of oxidation-transfer reactions performed using Se reagents is substantial; nevertheless, several new protocols are still published each year. Currently, the dominant trend in the field is to design greener and more environment-friendly protocols with a special emphasis on the use of organoselenium reagents as catalysts in the presence of milder cooxidants. This chapter summarizes the state of art in the field of Se-catalyzed oxofunctionalization reactions, focusing on the most recent reports.
The emergence of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an urgent public health crisis. Without available targeted therapies, treatment options remain limited for COVID-19 patients. Using medicinal chemistry and rational drug design strategies, we identify a 2-phenyl-1,2-benzoselenazol-3-one class of compounds targeting the SARS-CoV-2 main protease (Mpro). FRET-based screening against recombinant SARS-CoV-2 Mpro identified six compounds that inhibit proteolysis with nanomolar IC50 values. Preincubation dilution experiments and molecular docking determined that the inhibition of SARS-CoV-2 Mpro can occur by either covalent or noncovalent mechanisms, and lead E04 was determined to inhibit Mpro competitively. Lead E24 inhibited viral replication with a nanomolar EC50 value (844 nM) in SARS-CoV-2-infected Vero E6 cells and was further confirmed to impair SARS-CoV-2 replication in human lung epithelial cells and human-induced pluripotent stem cell-derived 3D lung organoids. Altogether, these studies provide a structural framework and mechanism of Mpro inhibition that should facilitate the design of future COVID-19 treatments.
Ebselen (EBS), 2-phenyl-1,2-benzisoselenazol-3(2H)-one, is an organoselenium pharmaceutical with antioxidant and anti-inflammatory properties. Furthermore, EBS is an excellent scavenger of reactive oxygen species. This property complicates conventional protocols for sensitizing and quenching reactive species due to potential generation of active intermediates that quickly react with EBS. In this study, the photochemical reactivity of EBS was investigated in the presence of (1) 1O2 and •OH sensitizers (Rose Bengal (RB), perinaphthanone, H2O2) and (2) reactive species scavenging and quenching agents (sorbic acid, isopropanol, sodium azide, tert-butanol) that are commonly employed to study photodegradation mechanisms and kinetics. The carbon analog of EBS, namely 2‐phenyl‐3H‐isoindol‐1‐one, was included as a reference compound to confirm the impact of the selenium atom on EBS photochemical reactivity. EBS does not undergo acid dissociation, but pH-dependent kinetics were observed in RB-sensitized solutions, suggesting EBS reaction with active intermediates (3RB2-*, O2⦁-, H2O2) that are not kinetically-relevant for other compounds. In addition, the observed rate constant of EBS increased in the presence of sorbic acid, isopropanol, and sodium azide. These findings suggest that conventional reactive species sensitizers, scavengers, and quenchers need to be carefully applied to highly reactive organoselenium compounds to account for reactions that are typically slow for other organic contaminants.
This book presents recent advances in and perspectives on the use of organoselenium compounds, primarily highlighting the new frontiers in the field of Green Chemistry, their therapeutic and biological relevance and new materials. Throughout its 200 pages, readers will find an updated and comprehensive review of new aspects of organoselenium chemistry and biochemistry. Fully referenced and written in an easy to read style, it offers readers a primary resource for including organoselenium derivatives in their projects.
This book will be of interest to specialists, students and researchers involved in a broad range of fields, from synthetic green chemistry to medicinal chemistry and the chemistry of natural products.
The connection between organoselenium compounds and green chemistry, despite having only recently emerged, is one of the subjects of this book. The first chapter highlights the use of Se-containing molecules as reagents and catalysts in new green protocols to access important organic transformations.
The book provides a wealth of examples of bioactive Se-containing molecules, especially focusing on those with potential therapeutic uses. The second chapter focuses on the state of the art concerning the role of organoselenium compounds as antioxidants, GPx mimics, and derivatives endowed with different bioactive properties. “Organoselenium in nature” is the title of the third chapter, which equips readers with essential information on the main natural organoselenium compounds and where they are found. Selected aspects of the metabolism of selenium in plants and microorganisms are also discussed. In closing, the book includes a chapter dedicated to recent advances concerning the nonbonding interactions between organochalcogen compounds. This is currently a hot topic in selenium chemistry and biochemistry, and here readers will find key insights into the chalcogen bond and its role in the biological activity of organoselenium compounds.
In the lay press, antioxidants are often regarded as magic compounds. Antioxidants are claimed to heal almost every human inconvenience. Chemically, an antioxidant is a compound which in relatively low concentration delays or prevents the oxidation of another compound. In other words, when two compounds are compared and one oxidises more easily than the other, the best oxidizable compound functions as the antioxidant for the other. This view takes away much of the magic that surrounds the antioxidant field. Just compare an easily oxidizable substance A with another molecule B that is rather difficult to oxidise and the compound A can be claimed to be an antioxidant. It does not come as a surprise therefore that many cardiovascular drugs have been characterized as antioxidants. Discussions on local concentrations that should be achieved in order for these drugs to be active as antioxidants pop up in the literature regularly. The notion that antioxidants function in a network of enzymatic and non-enzymatic antioxidants has also gained a lot of attention recently [1]. This antioxidant network differs between organs and its composition even differs at the cellular level. Interplay of pharmaceutical antioxidants with the physiological antioxidant network has not been widely investigated thus far. Rational use of pharmaceutical antioxidants however would require this particular knowledge [2].
Chronic stress or chronically high levels of glucocorticoids can result in memory impairment.
This study aimed to investigate if 4,4′-dichloro-diphenyl diselenide (p-ClPhSe)2 reverses memory impairment-related to stress caused by corticosterone administration in mice and its possible mechanism of action. Swiss mice received corticosterone (20 μg/ml) in their drinking water during four weeks. In the last week, the animals were treated with (p-ClPhSe)2 (1 or 5 mg/kg) by the intragastric route (i.g.) once a day for 7 days. The cognitive performance of mice was assessed through the object recognition test (ORT), the object location test (OLT) and the step-down passive avoidance test (SDPA), some of predictive tests of memory. Biochemical parameters were determined and locomotor activity of mouse was performed to gain insight in (p-ClPhSe)2 toxicity. The findings demonstrated that treatment with (p-ClPhSe)2 in both doses was effective in reversing memory deficits in the ORT, the OLT and the SDPA caused by corticosterone exposure in mice. Treatment with (p-ClPhSe)2 at both doses reversed the increase in the [³H] glutamate uptake by hippocampal slices of mice treated with corticosterone. By contrast, [³H] glutamate uptake by brain cortical slices was not altered in mice exposed to corticosterone. The Na⁺ K⁺ ATPase activity was not altered in hippocampus and cerebral cortices of mice treated with corticosterone. There was no sign of toxicity in mice treated with (p-ClPhSe)2. This organoselenium compound reversed memory impairment-related to stress caused by corticosterone and modulated hippocampal [³H]glutamate uptake in mice.
Selenium (Se) is an essential element to humans and animals. Since its diverse functions were discovered, its importance has been more and more revealed. After the discovery of selenium as a key component of many proteins, significant research efforts have been focused on its therapeutic and nutritional potentials and possible impacts on human and animal health. To date, 25 selenoprotein genes have been identified in humans and animals, where selenium exists in the form of an amino acid, selenocysteine (Sec). Sec incorporation into selenoproteins occurs through a unique process by recognizing UGA stop codon via the Sec-charged tRNA for delivering Sec into proteins. Most selenoproteins are antioxidant proteins and play critical roles in maintaining redox balance in living systems by removing reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). These reactive species are major threats to bio-macromolecules, such as DNAs, RNAs, proteins, lipids, and carbohydrates. They may cause molecular oxidation and DNA damage/mutation, alter gene expression patterns, weaken the immune system, and even lead to neurological disorders and other pathological conditions. As potential drugs and/or nutritional supplements, selenium-functionalized molecules (SeFMs) have shown benefits in preventing and/or treating many diseases, such as cancer, cardiovascular diseases, thyroid diseases, diabetes, AIDS, rheumatoid arthritis (RA), and neurodegenerative diseases. These promising results and therapeutic potentials of SeFMs have attracted tremendous attention from academia and pharmaceutical and biotech industries. However, further fundamental research and investigation are required to discover the ideal chemical, biochemical, and biological forms of selenium, appropriate dosages, and mechanisms of the selenium biological actions, since the tolerance window of selenium in humans may be limited.
Redox active molecules containing organoselenium or organotellurium groups catalyse the oxidation of cellular thiols by hydrogen peroxide and are currently being developed as therapeutic agents. Potentially these synthetic thiol peroxidase (TPx) mimics can protect cells from oxidative stress by catalysing the reduction of reactive oxygen species by the cellular thiol glutathione, an activity which mimics the function of the antioxidant enzyme glutathione peroxidase. Alternatively they can act as prooxidants by catalysing the oxidation of essential thiol species within the cell. However the structure activity relationships which determine the choice of thiol substrate, and hence the overall antioxidant or prooxidant outcome of drug administration, remain unknown. We report the first study that relates the pharmacological properties of TPx mimics with their solubility and catalytic activity using different thiol substrates. We used a series of structurally related compounds PhMCnH2n+1 (M=Se,Te; n=4-7) and investigated their ability to catalyse the oxidation of the cellular thiols glutathione and dihydrolipoic acid by hydrogen peroxide. The resulting rate constants (kobs) were then related to compound cytotoxicity and antioxidant versus prooxidant action in A549 cancer cells. The results show that the dihydrolipoic acid kobs values correlate with both cytotoxicity and prooxidant function. This enabled us to define a relationship, IC50=10+280e-5(DHLA kobs), which allows the prediction of TPx mimic cytotoxicity. In contrast, hydrophobicity and glutathione kobs were unrelated to the compounds’ redox pharmacology.
The generation of free radicals has been implicated in the pathologies of a number of cardiovascular diseases like infarction, stroke and atherosclerosis. Free radicals initiate lipid peroxidation, which leads to the impairment of membrane function and finally cell death. This often results in irrecoverable tissue damage. Therefore, potentially useful drugs would be those that not only have effects on the symptoms of the disease, but also demonstrate antioxidant and free radical scavenging properties. Some calcium entry blockers (CEBs) of the dihydropyridine (DHP) family (e.g. lacidipine, amlodipine, nifedipine) have been shown to possess radical scavenging activity in addition to their potent vasorelaxant properties. Some DHPs are active in inhibiting the autoperoxidation of rat cortical membranes. The order of potency was lacidipine>nimodipine>nifedipine, with lacidipine having an activity comparable to vitamin E. Lacidipine was also potent in protecting cells from the marked impairment of calcium homeostasis caused by oxidative stress induced by H2O2. Similarly, in the isolated rabbit heart electrolysis of the perfusion medium increases coronary artery pressure and this is antagonised by prior treatment with lacidipine. In vivo marked protection has been seen in animal models of vascular damage. In the stroke-prone spontaneously hypertensive rat lacidipine, at doses that do not block the development of hypertension, prevent mortality and tissue damage to brain and kidney, organs at risk in this model. Lacidipine also blocked the development of arterial lesions in two animal models of atherosclerosis : the hypercholesterolaemic rabbit and the hypercholesterolaemic hamster. These actions can be at least partially ascribed to mechanisms other than simple blood pressure reduction and might well relate to antioxidant activity.
The biochemistry of ‘reactive oxygen species’ (ROS) is an important field with practical implications, because whereas oxygen is an essential component for living organisms, the formation of reactive oxygen intermediates seems to be commonplace in aerobically metabolizing cells. In addition to aerobic metabolism-encompassing electron transfer chains and certain enzyme activities, environmental sources, such as air pollutants, photochemical smog, industrial chemicals, and ionizing radiation, as well as the metabolism of xenobiotics, contribute to the cellular steady-state concentration of ROS. Further, reactive species are formed as a response to diverse stimuli by specialized physiological reactions: the formation of oxyradicals during the respiratory burst and the release of the endothelium-derived releasing factor, identified as nitric oxide, are such examples.
Among the mammalian selenoenzymes, glutathione peroxidase (GPx) has attracted much attention due to its enormous importance in the defense against oxidative stress. The chemistry at the active site of GPx has been widely studied with the help of a number of small-molecule organoselenium compounds. Among these, ebselen (2-phenyl-1,2-benzisoselenazole-3(2H)-one) is currently undergoing phase III clinical trial for a number of disease states as it exhibits good antioxidant activity in vivo. However, the catalytic mechanism for the GPx activity of ebselen is not well established. In this article, different catalytic mechanisms proposed in the literature for the GPx-like antioxidant activities of ebselen and related selenenyl amides are described. Recently, it has been observed that ebselen and related compounds show poor catalytic activities in the presence of aryl or benzyl thiols as cofactors. The unusually low catalytic activities of these Sec-amide-substituted compounds have been ascribed to the undesired thiol exchange reactions that take place in the selenenyl sulfide intermediates due to the presence of strong Se...O nonbonded interactions. Furthermore, it has been shown that the extensive thiol exchange reactions can be prevented by using a dithiol as cofactor or by introducing S...N/O nonbonded interactions in selenenyl sulfide intermediates. In addition, it has been observed that the GPx activities of secamide-based compounds can be dramatically enhanced by the substitution at free-NH groups of the amide functionality that prevents the undesired thiol exchange reactions.
This chapter provides an introduction on the enzymatic system of defense. Next, it overviews the catalytic mechanisms of copper superoxide dismutase, manganese superoxide dismutase, and selenium glutathione peroxidase. Further, it talks about metal-based synthetic antioxidants and nonmetal-based antioxidants. The chapter concludes with a discussion on nitrones that were designed as spin traps for detecting transient free radicals using electron paramagnetic resonance. Among the family of nitrones, two classes have been widely employed, the cyclic nitrones derived from the 5,5-dimethyl-1- pyrroline-N-oxide (DMPO) and the linear ones derived from the a-phenyl-N- tert -butyl nitrone (PBN). This section on nitrones discusses the protective, antiaging and neuroprotective effects of nitrones.
Cyclic seleninate esters function as mimetics of the antioxidant enzyme glutathione peroxidase and catalyze the reduction of hydrogen peroxide with a stoichiometric thiol. While a single electron-donating methoxy substituent para to the selenium atom enhances the catalytic activity, m-methoxy groups have little effect and o-methoxy substituents suppress activity. The effects of multiple methoxy groups are not cumulative. This behavior can be rationalized by opposing mesomeric and steric effects. Oxidation of the product disulfide via its thiolsulfinate was also observed.
Catalytic activity of a series of potent amide- and amine-based organoselenium compounds are studied in the presence of various aromatic thiols having electron donating and electron withdrawing substituents on the phenyl ring. This study suggests that the antioxidant activities of the synthetic GPx mimics can be significantly increased by the incorporation of a suitable electron donating group on the phenyl ring of an aromatic thiol.
Selenium plays a key role in biology by mediating many important physiological processes. In proteins, selenium is present in the form of selenocysteine (Sec, U), which is generally considered as the 21st amino acid. Although Sec is a homolog of cysteine (Cys, C) and has similar structural and functional properties, the reactivity of Sec residues in proteins is significantly different from that of Cys due to the low redox potential and high nucleophilicity of Sec. In this chapter, some of the well-studied examples of selenoenzymes and their synthetic mimics are discussed. Synthetic compounds that mimic the function of glutathione peroxidase (GPx) and iodothyronine deiodinase (ID) are used extensively to understand the chemical mechanism of the enzymes. The chemistry of Sec residue at the active site of GPx is widely studied with the help of a number of small-molecule organoselenium compounds. Ebselen was the first synthetic GPx mimic to undergo clinical studies for a number of disease states, including stroke, inflammation, and hearing loss, although ebselen and its analogues exhibit poor GPx activity in the presence of thiols due to undesired thiol-exchange reactions. The GPx activity of several tert-amine-based diselenides is found to be much higher than that of ebselen and related compounds. In these compounds, the amine moiety plays an important role in modulating the reactivity of different intermediates involved in the catalytic cycle. In addition to selenenyl amides and diaryl diselenides, a number of spirodiazaselerunanes are studied as GPx mimics. The role of Sec in iodothyronine deiodinases, which control the thyroid hormone levels, is also discussed in this chapter. Recently, some naphthyl-based selenols and thiols are reported as mimics of deiodinases.
Keywords:
antioxidants;
ebselen;
glutathione peroxidase;
iodothyronine deiodinase;
selenium;
selenoenzymes;
selenophosphate synthetase;
thioredoxin reductase
Ebselen is a synthetic, lipid-soluble seleno-organic compound. The high electrophilicity of ebselen enables it to react with multiple cysteine residues of various proteins. Despite extensive research on ebselen, its target molecules and mechanism of action remains less understood. We performed biochemical as well as in vivo experiments employing budding yeast as a model organism to understand the mode of action of ebselen. The growth curve analysis and FACS (florescence activated cell sorting) assays revealed that ebselen exerts growth inhibitory effects on yeast cells by causing a delay in cell cycle progression. We observed that ebselen exposure causes an increase in intracellular ROS levels and mitochondrial membrane potential, and that these effects were reversed by addition of antioxidants such as reduced glutathione (GSH) or N-acetyl L-cysteine (NAC). Interestingly, a significant increase in ROS levels was noticed in gdh3-deleted cells compared to wild-type cells. Furthermore, we showed that ebselen inhibits GDH function by interacting with its cysteine residues, leading to the formation of inactive hexameric GDH. Two-dimensional gel electrophoresis revealed protein targets of ebselen including CPR1, the yeast homologue of Cyclophilin A. Additionally, ebselen treatment leads to the inhibition of yeast sporulation. These results indicate a novel direct connection between ebselen and redox homeostasis.
The oxidation of allyl selenide 12 with hydrogen peroxide produced the corresponding allyl selenurane 14, the cyclic seleninate ester 4 or the rearranged O-allyl seleninate ester 18, dependng on the conditions. Crossover experiments with selenide 12 and its deuterated crotyl analog 27 indicated an intramolecular rearrangement that proceeds by an intramolecular pathway where the allyl or crotyl group is translocated via its distal carbon atom to the hydroxymethyl functionality. VT-NMR experiments with cyclic seleninate ester 4 revealed fluxional behavior at room temperature that was catalyzed by trifluoroacetic acid. Computational studies indicated an activation energy of 12.3 kcal mol-1 for hydroxyl interchange at selenium, comparable to the value of 15.5 kcal mol-1 derived from the NMR experiments. The glutathione peroxidase-like activity of 4 was measured in an assay where the catalysis of the reduction of hydrogen peroxide with benzyl thiol was monitored by the appearance of dibenzyl disulfide. The catalytic activity of 4 was double that observed with the unsubstituted seleninate ester 2, but was limited by the competing accumulation of the relatively inert selenenyl sulfide 32, resulting in a deactivation pathway that competes with the primary catalytic cycle.
This thesis focuses on the possible health-beneficial effects of the
flavonoid quercetin. Especially the relation between the in vitro and in vivo
behavior of quercetin is explored. Furthermore, the possible use of this
flavonoid in sarcoidosis, an inflammatory disease, is examined.
Firstly, the behavior of quercetin during oxidative stress was evaluated
in vitro. Chapter 2 describes the protection against lipid peroxidation
offered by catechol-containing antioxidants such as quercetin. The effects of
its oxidation products, formed during this protection, are discussed. In
chapter 3 the reaction of the main oxidation product of quercetin, i.e. QQ,
with thiols is further examined. Especially the reactions of QQ with GSH or
with vitamin C are evaluated and compared. To investigate the reaction of
QQ with DT-diaphorase (NQO-1), the experiments described in chapter 4 are
performed. The possible role of this enzyme in the protection against QQinduced toxicity is examined. Since the QQ-toxicity appears to be focused on
its reaction with thiols, chapter 5 focuses on the fate of the adducts formed
between QQ and various thiols, including GSH, the most abundant
endogenous thiol. The in vitro work was concluded by investigating the
protective effect of quercetin in relation to the potential toxic effects of its
oxidation products in a more integrated cell system, using rat lung epithelial
cells (RLEs) as described in chapter 6.
Secondly, the in vitro and ex vivo anti-inflammatory as well as the in
vivo anti-oxidative effects of quercetin have been examined. Chapter 7
presents the effects of a four-weeks comprising quercetin intervention on the
antioxidant status as well as on the basal and ex vivo-induced inflammation
in healthy volunteers. The potential toxicity of QQ is also discussed. The ex
vivo anti-inflammatory effect of quercetin has further been examined in both
sarcoidosis patients and healthy controls in chapter 8. Moreover, the
antioxidant status of both the patients and their matched controls has been
quantified.
Thirdly, the in vivo anti-oxidative and anti-inflammatory effects of
quercetin supplementation in sarcoidosis patients are reported in chapter 9.
Implications for the use of antioxidant supplementation, with for example
quercetin, in the treatment of this disease are given.
Finally, the results and impacts of our findings are summarized in
chapter 10. Implications for further research are also given.
This chapter discusses the reaction of lipoic acid with ebselen and hypochlorous acid. Lipoic acid plays a pivotal role in energy metabolism. Its antioxidant activity, however, is also recognized in normal and pathological conditions. In vivo, lipoic acid shuffles between its reduced [dihydrolipoic acid or 6,8-dimercaptooctanoic acid, L(SH)2] and oxidized state (lipoic acid or 1,2-dithiolane-3-pentanoic acid). Dihydrolipoic acid is characterized by two thiol groups per molecule; these thiol groups provide the compound with its good antioxidant efficacy. In this regard, its role in the glutathione (GSH) peroxidase-like activity of the small molecule ebselen is interesting. Ebselen is an organoselenium compound with antiinflammatory activity. It catalyzes the reduction of peroxides by GSH. Replacing GSH by L(SH)2 improves the peroxidase-like activity of ebselen. In the oxidized state of lipoic acid, the two sulfur atoms are attached to each other as part of a five-membered 1,2-dithiolane ring. The strain in this ring is responsible for a unique reactivity. An example of this reactivity is the ability of this molecule to scavenge hypochlorous acid (HOCl). By scavenging the neutrophil oxidant HOCl, the elastase inhibitor αl-antiproteinase (API) is protected from oxidation. The reaction scheme for the catalysis of the GSH peroxidase reaction by ebselen is analogous to that of the enzyme.
This chapter attempts to narrow the gap where antioxidant pharmacology meets with organic chemistry. It is intended to serve as a reference to substances that have been described, designed, or developed as therapeutically relevant antioxidants. It is obvious that the present contribution cannot cover more than a limited number of examples of antioxidants from the large variety of structural classes that have been studied as possible drug candidates. For this purpose, it most fruitful to cover compounds that have emerged from in vitro experimentation and structure–activity studies. The chapter discusses antioxidant effects in molecular and structural terms. The synthetic compounds have been classified chemically, rather than by therapeutic area, as the latter, in most instances, is a matter of the preference of the researchers rather than inherent properties of the substances. Some ranges regarding dose or concentration have been included to indicate the approximate potencies of compounds, especially when standard in vitro assays have been utilized. The enzyme systems that are devoted to detoxification of reactive and potentially pathological oxygen metabolites have also been thoroughly treated by others, and will be mentioned to provide a basis for the discussion of synthetic enzyme mimics. The chapter attempts to present the various conceivable approaches that are at hand for attenuating oxidative tissue injury and the harmful effects that are ascribed to free radicals.
Synthesis of 2-Phenyl-1,2-benziso[77Se]selenazol-3(2H)-one has been accomplished in one step from 94% 77Se and commercially available starting materials in 76% yield based on the enriched elemental selenium.
Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a lipid-soluble organoselenium compound, exhibits numerous biological activities both in vitro and in vivo systems. This compound is undergoing clinical trials for a number of disease states such as stroke and hearing loss. It is known that ebselen exhibits glutathione peroxidase activity (GPx) and is a remarkable scavenger of reactive oxygen species (ROS) such as peroxynitrite (PN). The rate of the reaction between ebselen and PN has been shown to be about three orders of magnitude higher than that of naturally occurring small molecules, such as cysteine, methionine and ascorbate. It is also known that ebselen and related compounds effectively protect against lipid peroxidation induced by transition metal ions. However, the mechanism by which ebselen exerts its antioxidant activity and the importance of the cyclic selenazole moiety are still not well-understood. In this article, the complex chemical mechanisms involved in the antioxidant activity of ebselen and related compounds are discussed.
The interaction of the novel antioxidant drug ebselen, 2-phenyl-1,2 benzisoselenazol-3 (2H)-one, with human serum albumin (HSA) was investigated by spectrophotometry. 6.7 mM, pH 7.4 of phosphate buffer containing 0.4 % (v / v) dimethylformamide (DMF) was used as the solvent. Various amounts of ebselen and HSA mixtures were used. Incubations of the mixtures were performed for 15 minutes at 25°C and 30 minutes at 37°C. An irregular change and the lack of an isobestic point in the final spectra of the mixtures indicated that the formation of mixed albumin selenodisulfides. It was also determined that the structures of the final products formed from the interactions of the drug and HSA, were depended on both drug and HSA concentrations. Keywords: Ebselen (PZ 51), human serum albumin, spectrophotometry. Ebselenin İnsan Serum Albüminine Bağlanmasının Spektrofotometrik olarak İncelenmesi Antioksidan özellikte yeni bir ilaç maddesi olan ebselen'in (2-phenyl-1,2 benzisoselenazol-3 (2H)-one) insan serum albümini (HSA) ile etkileşimi spektrofotometrik olarak incelendi. Çözücü olarak 0.4 % (v / v) dimetilformamid (DMF) içeren 6.7 mM, pH 7.4 fosfat tamponu ve farklı miktarlarda ebselen-HSA karışımları kullanıldı. Karışım inkübasyonları 25°C de 15 dakika ve 37°C de 30 dakikada gerçekleştirildi. Sonuç spektrumlarındaki düzensiz bir değişim ve bu spektrumlarda izobestik bir noktanın bulunmayışı karışık albümin selenodisülfürlerin oluştuğunu göstermiştir. Ayrıca ebselen ile HSA etkileşimi sonucunda oluşan ürün yapılarının ilaç ve HSA'nın her ikisinin de konsantrasyonlarına bağlı olduğu saptanmıştır. Anahtar kelimeler: Ebselen (PZ51), insan serum albümini, spektrofotometri.
Synchrotron radiation induced X-ray emission (SRIXE) spectroscopy was used to map the cellular uptake of the organoselenium-based antioxidant drug ebselen using differentiated ND15 cells as a neuronal model. The cellular SRIXE spectra, acquired using a hard X-ray microprobe beam (12.8-keV), showed a large enhancement of fluorescence at the K(α) line for Se (11.2-keV) following treatment with ebselen (10 μM) at time periods from 60 to 240 min. Drug uptake was quantified and ebselen was shown to induce time-dependent changes in cellular elemental content that were characteristic of oxidative stress with the efflux of K, Cl, and Ca species. The SRIXE cellular Se distribution map revealed that ebselen was predominantly localized to a discreet region of the cell which, by comparison with the K and P elemental maps, is postulated to correspond to the endoplasmic reticulum. On the basis of these findings, it is hypothesized that a major outcome of ebselen redox catalysis is the induction of cellular stress. A mechanism of action of ebselen is proposed that involves the cell responding to drug-induced stress by increasing the expression of antioxidant genes. This hypothesis is supported by the observation that ebselen also regulated the homeostasis of the transition metals Mn, Cu, Fe, and Zn, with increases in transition metal uptake paralleling known induction times for the expression of antioxidant metalloenzymes.
Ebselen (1), the quintessential mimic of the antioxidant selenoenzyme glutathione peroxidase (GPx), is a potential chemopreventative for various diseases associated with oxidative stress. Density-functional theory (DFT) and solvent-assisted proton exchange (SAPE) are used to model the complex mechanism for scavenging of reactive oxygen species by 1. SAPE is a microsolvation method designed to approximate the role of bulk solvent in chemical processes involving proton transfer. Consistent with experimental studies, SAPE studies predict the reaction of 1 with thiol (RSH) to form a selenenyl sulfide 2 to be preferred under most conditions, with an alternate pathway through a selenoxide 3 possible at high reactive oxygen species (ROS) concentrations ([ROS] ≫ [RSH]). The reduction of 2 to the selenol 4, known to be rate-determining in the protein, has a high SAPE activation barrier due to a strong Se···O interaction which reduces the electrophilicity of the sulfur center of the -SeS- bond of 2. Thiols, such as dithiols and peptide-based thiols, are expected to overcome this barrier through structural features that increase the probability of attack at this sulfur. Thus, in vivo, the GPx-like pathway is the most likely mechanism for 1 under most circumstances, except, perhaps, under extreme oxidative stress where initial oxidation to 3 could compete with formation of 2. Simple thiols, used in various in vitro studies, are predicted by SAPE modeling to proceed through oxidation of 2 to a seleninyl sulfide intermediate. Overall, SAPE modeling provides a realistic interpretation of the redox mechanism of 1 and holds promise for further exploration of complex aqueous-phase reaction mechanisms.
Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl-, Br- and SCN- by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×10(3)-5.8×10(6) M-1·s-1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M-1·s-1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.
Conformationally restricted naphthalene peri-diselenides function as glutathione peroxidase mimetics with superior catalytic activity to that displayed by typical acyclic diaryl diselenides. Their activity was increased by electron-donating methoxy substituents, while a further 100-fold increase was observed with the corresponding ditelluride.
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