Ebselen: A substrate for human thioredoxin reductase strongly stimulating its hydroperoxide reductase activity and a superfast thioredoxin oxidant

Abstract

Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], a selenoorganic compound with glutathione peroxidase-like activity is used in clinical trials against stroke. Human and bovine TrxR catalyzed the reduction of ebselen to ebselen selenol by NADPH with an apparent K M-value of 2.5 M and a kcat of 588 min ؊1 . The addition of thioredoxin (Trx) stimulated the TrxR-catalyzed reduction of ebselen several-fold. This result was caused by a very fast oxidation of reduced Trx by ebselen with a rate constant in excess of 2 ؋ 10 7 M ؊1 s ؊1 . This rate is orders of magnitude faster than the reaction of dithiol Trx with insulin disulfides. Ebselen competed with disulfide substrates for reduction by Trx and, therefore, acted as an inhibitor of protein disulfide reduction by the Trx system. The inherent H2O2 reductase activity of mammalian TrxR dependent on its active-site selenocysteine residue was stimulated 10-fold by 2 M ebselen and 25-fold in the additional presence of 5 M Trx. Furthermore, the apparent KM-value of TrxR for H2O2 was lowered 25-fold to about 100 M. Our results demonstrate that ebselen is a TrxR peroxidase which, in the presence of Trx, acted as a mimic of a peroxiredoxin. The activity with TrxR and oxidation of reduced Trx offer mechanistic explanations for the in vivo effects of ebselen as an antioxidant and anti-inflammatory agent. Our results demonstrate that the mechanism of action of ebselen may be predominantly via the Trx system rather than via glutathione.selenol ͉ inflammation ͉ signal transduction ͉ oxidative stress ͉ redox regulation E bselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] is a lipidsoluble seleno-organic compound that exhibits a weak glutathione peroxidase (GPx)-like activity in vitro (1-6). The specificity for substrates ranges from H 2 O 2 to smaller organic hydroperoxides and includes membrane-bound phospholipid and cholesterylester hydroperoxides. Because ebselen is effective against membrane hydroperoxides, it inhibits both nonenzymatic and enzymatic lipid peroxidation in cells and has anti-inflammatory activity in various animal models (5, 6). Ebselen also will directly inhibit inflammation-related enzymes such as 5-lipoxygenase, nitric oxide synthases, NADPH oxidase, protein kinase C, and ATPase by chemically modifying an SH-group forming a selenosulfide complex (5, 6).Because of its anti-inflammatory properties, ebselen has been used in the treatment of patients with acute ischemic stroke (7,8) or delayed neurological deficits after aneurysmal subarachnoid hemorrhage (9). The results of these clinical trials indicate the benefit of ebselen as a neuroprotective agent. Also, recent animal studies show neuroprotective, antioxidant, and antiinflammatory actions of ebselen in a rodent model of permanent middle as well as transient cerebral artery occlusion (10-13).Thioredoxin (Trx) reductase (TrxR) is a dimeric FADcontaining enzyme that catalyzes the NADPH-dependent reduction of the active-site disulfide in oxidized Trx (Trx-S 2 ) to give a dithiol in reduced Trx [Trx-(SH) 2 ] (14...

2-Methoxyestradiol induces apoptosis in Ewing sarcoma cells through mitochondrial hydrogen peroxide production

“…This interpretation is based on the rise in intracellular H 2 O 2 level in 2-Metreated EW7 cells and on the marked protection against apoptosis exerted by Ebselen, (Sies, 1994;Zhao et al, 2002) an antioxidant that mimics glutathione peroxidase activity by reducing intracellular accumulation of H 2 O 2 . Furthermore, rotenone, an inhibitor of the mitochondrial respiratory chain, prevented 2-Me-induced apoptosis and H 2 O 2 production, thus implicating the mitochondrial electron transport chain as the source of H 2 O 2 generation.…”

“…To test this possibility, we examined the effect of distinct antioxidants/ROS scavengers on 2-Me-induced apoptosis in EW7 cells. The following antioxidants were used: N-acetyl-l-cysteine (Nac) (Curtin et al, 2002), which is readily taken up by cells and is rapidly converted to glutathione (GSH); vitamin E (Halliwell and Gutteridge, 1984), which is a well-known inhibitor of lipid peroxidation; Tiron (Ledenev et al, 1986), which is a cell-permeable O 2 À scavenger; and Ebselen (Sies, 1994;Zhao et al, 2002), a seleno-organic component that mimics the activity of the glutathione-peroxidase enzyme by converting H 2 O 2 to H 2 O. As shown in Figure 5a, pretreatment of cells with Ebselen completely prevented 2-Me-induced 2-Me-induced apoptosis and reactive oxygen species M Djavaheri et al apoptosis as assessed by nucleosome DNA fragmentation assay, whereas Nac and Tiron had no effect.…”

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“…Recent animal studies also show neuroprotective, antioxidant, and anti-inflammatory actions of ebselen in a rodent model of transient cerebral artery occlusion (84,154). Recently, ebselen was shown to be an excellent substrate for mammalian TrxR and a superfast oxidant of reduced Trx (352). Therefore, ebselen is mainly a peroxiredoxin mimic, and its target is the thioredoxin system (351,352).…”

“…Thus, TrxR participates in selenium metabolism via selenium-selenium interactions (115) and plays an important role in controlling selenoprotein synthesis. In addition, TrxR can directly reduce lipid hydroperoxides and hydrogen peroxide, and the presence of selenium compounds such as selenocysteine or ebselen strongly increases this activity (37,352). This was intriguing when originally observed in studies of selenite reduction by mammalian TrxRs, before it was known that the enzyme was a selenoenzyme.…”

“…Recently, ebselen was shown to be an excellent substrate for mammalian TrxR and a superfast oxidant of reduced Trx (352). Therefore, ebselen is mainly a peroxiredoxin mimic, and its target is the thioredoxin system (351,352). Ebselen is reduced to the selenol by TrxR or Trx and reoxidized by peroxides, including hydrogen peroxides.…”