Ebselen, a Seleno-organic Antioxidant, as an Electrophile

Abstract

Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], a seleno-organic compound showing glutathione peroxidase-like activity, is one of the promising synthetic antioxidants. In the present study, we investigated the electrophilic potential of this antioxidant and established the mechanism of the cysteine-targeted oxidation of protein. In addition, using ebselen as an electrophilic probe, we characterized the cysteine residues required for posttranslational modification into an electrophile sensor protein in the phase 2 detoxification response. Ebselen showed a potent antioxidant effect against the spontaneous and 4-hydroxy-2-nonenal-stimulated production of intracellular reactive oxygen species in rat liver epithelial RL34 cells. Meanwhile, upon in vitro incubation with a redox-active sulfhydryl protein (thioredoxin), ebselen showed a strong electrophilic potential of mediating the formation of selenenylsulfide and intra- and intermolecular disulfide linkages within the protein. By taking advantage of this antioxidant and electrophilic property of ebselen, we characterized posttranslational modification of Kelch-like ECH-associated protein 1 (Keap1), an electrophile sensor protein, which represses the ability of the transcription factor NF-E2-related factor 2 (Nrf2) upon induction of the phase 2 detoxification response. Ebselen potently induced the gene expression of a series of phase 2 enzymes in rat liver epithelial RL34 cells, which was associated with the formation of a high molecular weight complex of Keap1. Furthermore, a cysteine residue in Keap1, C151, was found to be uniquely required not only for the formation of the complex but also for the induction of the phase 2 response by ebselen. Thus, this unique antioxidant and electrophilic property of ebselen giving rise to the cysteine-targeted oxidation enabled us to evaluate the role of sensor cysteines in redox regulation of protein function under electrophile stress.

Acrolein: Sources, metabolism, and biomolecular interactions relevant to human health and disease

“…It appeared that the reactivities of the cysteines in human Keap1 are different from the corresponding cysteines in mouse Keap1 [112][113][114][115]117]. Despite some uncertainty regarding the exact order of the reactivity of the cysteine residues towards different electrophiles, Cys-151 (located in the BTB domain) was identified as the most reactive nucleophilic site in human Keap1 [113,117]. It is conceivable that multiple cysteines in Keap1 can be modified by different inducers and it is likely that sites of sulfhydryl modification may vary among the different chemical compound classes and across species [118].…”

Chemistry of the Cysteine Sensors in Kelch-Like ECH-Associated Protein 1

“…Ebselen, 31, has variously been reported as a phase 2 enzyme inducer that acts through the Nrf2 pathway (50). It has been shown to decrease basal ROS and the flux induced by 4-hydroxynonenal.…”

Sites of alkylation of human Keap1 by natural chemoprevention agents

“…Regardless, these results indicate C151 is extremely reactive toward these three chemoprevention agents. Located in the BTB domain of Keap1, sitedirected mutagenesis studies have indicated that C151 is essential for inhibition of Keap1-dependant ubiquitination and degradation of Nrf2 in response to all tested agents, including tBHQ and sulforaphane [3], NEPP11, an endogenous neurite outgrowth-promoting prostaglandin [34], and ebselen, a seleno-organic drug [35]. Taken together, C151 plays a critical role in the Keap1-Nrf2 signaling system, and our results support the view that C151 is a target for Nrf2 activation by Michael addition reaction acceptors that function as ARE inducers.…”