Ebselen as a Glutathione Peroxidase Mimic: Alias Chemical Chameleon Antioxidant

Ebselen (2-phenyl-1, 2-benzisoselenazol-3 (2H)-one) is a glutathione peroxidase mimic that catalyzes the reduction of a hydroperoxide with the oxidation of a thiol. In vitro models indicate that the oxidative protection ebselen affords to cells is due to its glutathione peroxidase (GPx) mimic activity. This paper will describe ebselen’s reactions, detection, and pharmacological potential. Introduction Glutathione peroxidase was discovered over 40 years ago in erythrocytes [11]. Around the same time, nutrition studies found selenium to be an important micronutrient. Selenocysteine is now known to be a required component of the active site of GPx [7]. Four versions of glutathione peroxidase have been found (classical erythrocyte GPx-I [11], PhGPx [24, 23], S. S. Peters Ebselen 3 plasma GPx [22,21], and GI-GPx) along with many selenopeptides and selenoproteins with unknown functions [Behne]. GPx-I is a tetramer with four active sites [6,26]. Ebselen ( PZ 51) was first described in 1984 as a selenoorganic molecule with glutathione peroxidase mimic activity [13,25]: (A) (B) Figure 1. Glutathione peroxidase (Athe peptide backbone of a bovine GSH peroxidase subunit) [5] and ebselen (Bthe synthesized drug) [7] structures are shown to make their size and complexity differences apparent. The structural differences in these compounds contribute to ebselen's low specificity in comparison to GPx. Fewer conformational folds and an absence of amino acid residue charges frees ebselen from the multiple binding requirements of GSH peroxidase. The molocules’ active sites, however, have been shown to have closely related mechanistic functions. An S. S. Peters Ebselen 4 understanding of ebselen chemistry and biochemistry clearly depends upon an understanding of glutathione peroxidase reactivity which will be elucidated in the following section. GPx Reactivity Three steps are considered to be involved in the enzymatic catalysis of GPx and PhGPx [5]. The active site for both of these molecules is a selenocysteine group [19]. The enzymebound selenocysteine reacts with an alkyl hydroperoxide to produce selenic acid and an alkyl hydroxide in reaction (1) [19]. In reaction (2), the selenic acid reacts with glutathione to produce selenosulfide (refered to as selenodisulfide in the reference) and water [19]. Reaction (3) transforms selenodisulfide back into the origional enzyme-bound selenocysteine through its reaction with another glutathione which also results in the formation of glutathione disulfide, GSSG [19]. In the stoichiometric reaction (4), the sum of reactions (1), (2), and (3) is illustrated [19]. E-Cys-SeH + ROOH à E-Cys-SeOH + ROH (1) E-Cys-SeOH + GSH à E-Cys-Se-SG + H2O (2) E-Cys-Se-SG + GSH à E-Cys-SeH + GSSG (3) Net: ROOH + 2GSH à ROH + H2O + GSSG (4) S. S. Peters Ebselen 5 GPx Mimic Reactivity A kinetic study [9] indicates that the above reaction can be catalyzed by ebselen through an analogous reaction scheme [19,5,27,25]. In this scheme (Figure 2), the ebselen selenol moiety is considered to be primarily responsible for the GPx activity of the GPx mimic [12]. Ebselen can use thiols other than GSH as substrates in this reaction (i.e. dithioerythretol [13], Nacetylcysteine [2], and dihyrolipoate [19]). Ebselen’s relatively low enzymatic binding specificity in comparison to GPx is demonstrated through these examples [19]. The difference is at least partially due to ebselen’s less complicated structure and smaller size. Figure 2. Ebselen shows a directly analogous reaction scheme to glutathione peroxidase. Thiols and hydroperoxides react with ebselen and it’s metabolites in recycling loops (A-D). Primary compounds of interest are ebselen (I), ebselen selenoxide (II), ebselen selenenylsulphide (III), ebselen selenol (IV), and ebselen diselenide (V). Symbols include R (residues of polyunsaturated phospholipids, cholesteryl esters, cholesterol or any other organic hydroperoxide or hydrogen) and R’ (glutathione, dihydrolipoate, Nacetylcysteine or thiol protein residue). Excess hydroperoxide over thiol favors cycle A. Cycles B, C, and D are preferred in other circumstances. Aprotic solvent environments favor cycle B, where aqueous systems favor a proportion of C and D dependant on the types of thiols present [17]. S. S. Peters Ebselen 6 Table 1. Selenol’s role as a primary factor in the glutathione peroxidase activities of ebselen can be further demonstrated by looking at it’s relatively high reactivity toward hydroperoxides with these second-order rate constants [17]. GPx Mimic Activity Detection Coupled enzymatic assays, GSH removal assays, and hydroperoxide removal assays have all been used in the detection of GPx mimic activity [19]. The coupled enzymatic assay utilizes a ratelimiting test reaction with GSSG. NADPH loss is followed via absorbance spectrophotometry [13,9]. The GSH removal assay stops the reaction, then tests for remaining GSH via thionitrobenzoate formation with the addition of Ellman’s reagent [25] or the formation of a monobromobimane adduct [2]. A typical version of the hydroperoxide removal assay utilizes the iron-thiocyanate compex [2,3] for GPx mimic activity detection. Direct radical scavenging activity The discovery of ebselen’s radical scavenging abilities was made with pulse radiolysis studies following selenoorganic reactions with 1,2-dichloroethane radicals and halogenated peroxyl radicals [18]. The reaction of trichloromethyl peroxyl radicals and ebselen produced a rate constant (Table 2) within the same range as alpha-tocopherol (vitamin E) in similar circumstances [19, 18]. In spite of this, biological model systems using competition kinetics showed that ebselen radical scavenging was not effective [19, 10, 16]. One analysis was based on the inhibition of crocin carotinoid bleaching by hydroperoxyl radicals (Table 3) [10] S. S. Peters Ebselen 7 Table 2. An antioxidant rate constant comparison upon reaction with trichloromethyl peroxyl radicals shows the relatively high activity of ebselen [18]. Table 3. Radical scavenging activity is indicated by Ka/Kc (the ability of molecules to interact with hydroperoxyl radicals). Ka indicates the rate constant for antioxidant/free radical interactions. Kc indicates the rate constant for crocin/free radical interactions. Higher values indicate more interactions and, therefore, more radical scavenging activity [10]. Pharmacological potential resulting from the GPx mimic’s prescence in vivo Glutathione peroxidase mimics provide antioxidant protection against lipid peroxidation and are metabolized without evidence of significant toxicity [19]. The reduction of lipid hydroperoxides present in liposomes or lipoproteins is proposed as the mechanism leading to this protection [9,10]. As a more specific example, ascorbate and NADPH have been used in vitro as reductants with ebselen to protect against ironinduced lipid peroxidation [13, 8, 15]. Ebselen’s role as an antioxidant is primarily based on its activity as a GPx mimic, and especially its role as a PhGPx, phospholipid hydroperoxide glutathione peroxidase, mimic [19, 10, 16]. The hydrophobicity of ebselen contributes to its effectiveness in antioxidant activity in and around membranes while its small size gives it the ability to diffuse and protect cellular compartments that are inaccessible to GPx [19]. Molecules from the ebselen catalytic cycles are eventually broken down and their products, including 4’-Glucuronyloxy-2-methylselenobenzanilide and 2(Methylseleno)-benzoic acid are excreted in the urine, without any evidence of toxic side effects [19, 4]. In low concentrations, ebselen has been shown to inhibit enzymes involved in S. S. Peters Ebselen 8 inflammation (lipoxygenases, NO synthetases, NADPH, oxidase, protein kinase C, and H/ KATPase ) [17]. Human cell experiments involving alimentary deficiency symptoms, which were found to be selenium-responsive, give further indirect evidence of ebselen’s antioxidant benefits as they relate to it’s selenium dependance [5]. A few symptoms which showed a positive response were: impaired hexose monophosphate shunts (erythrocytes), thrombasthenia (thrombocytes), slightly impared killing (neutrophiles), weakness/myalgy (skeletal muscles), and Keshan disease (heart muscles) [5]. SummaryThe role of antioxidants in maintaining a primarily reducing environment withinbiological systems is supported through well characterized chemical reactivity in vitro. Themechanisms of ebselen metabolism pathways and transport in vivo are not well understood andthere is still much work to be done in other areas, such as long term toxicity due to ebselenexposure, before clinical trials can be considered. Glutathione peroxidase mimic’s flexability insubstrate binding, combined with it’s accessability throughout the cellular milieu, and a lack ofsignificant toxicity could make it a potentially powerful and versatile therapeutic antioxidant. References1. Behne D, Hilmert H, Scheid S, Gessner H, Elger W. (1988) Evidence for specific seleniumtarget tissues and new biologically important selenoproteins. Biochim Biophys Acta. 966:12-21.2. Cotgreave IA, Moldeus P, Brattsand R, Hallberg A, Andersson CM, Engman L. 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