The Inhibition of Mammalian 15-Lipoxygenases by the Anti-Inflammatory Drug Ebselen: Dual-Type Mechanism Involving Covalent Linkage and Alteration of the Iron Ligand Sphere

Abstract

Mammalian lipoxygenases have been implicated in inflammation and atherosclerosis and, thus, lipoxygenase inhibitors may be of pharmacological interest. In cells, lipoxygenases occur in a catalytically silent ground state that requires activation to become active. We found that the seleno-organic drug ebselen [2-phenyl-1, 2-benzisoselenazol-3(2H)-one], which exhibits anti-inflammatory properties, irreversibly inhibited pure rabbit 15-lipoxygenase, with an IC50 in the nM range when preincubated with the enzyme in the absence of fatty acid substrates. Subsequent dialysis, gel filtration, or substrate addition did not restore the enzyme activity, and experiments with [14C]ebselen indicated a covalent linkage of the drug. The presence of sulfhydryl compounds in the incubation mixture prevented both enzyme labeling and inactivation, but we did not see any reactivation when sulfhydryl compounds were added afterward. X-ray absorption studies indicated that ebselen did alter the geometry of the iron ligand sphere, and the data are consistent with an iron complexation by the drug. When fatty acid substrate was present during lipoxygenase-ebselen interaction, the inhibitory potency was strongly reduced and a competitive mode of action was observed. These data suggest that ebselen inactivated the catalytically silent ground-state lipoxygenase irreversibly by covalent linkage and alteration of the iron ligand sphere. In contrast, it functions as a competitive inhibitor of the catalytically active enzyme species. The pharmacological relevance of ebselen as a potential in vivo lipoxygenase inhibitor will be discussed.

11,12,15-Trihydroxyeicosatrienoic acid mediates ACh-induced relaxations in rabbit aorta

“…In previous studies, we described inhibitors of each of these biosynthetic steps. The 15-lipoxygenase is inhibited by CDC, NDGA, or ebselen, whereas miconazole and clotrimazole inhibit the hydroperoxide isomerase (26,27,32).…”

“…Similar studies were performed in vessels pretreated for 10 min with the phospholipase A2 inhibitors mepacrine (10 Ϫ5 M) and 7,7-dimethyl-5,8-eicosadienoic acid (DEDA, 1.5 ϫ 10 Ϫ5 M), the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-␣-cyanocinnamate (CDC, 10 Ϫ6 M), nordihydroguaiaretic acid (NDGA, 5 ϫ 10 Ϫ5 M), and ebselen (2.5 ϫ 10 Ϫ5 M), or the hydroperoxide isomerase inhibitors miconazole (2 ϫ 10 Ϫ5 M) and clotrimazole (5 ϫ 10 Ϫ5 M) (26,27,32). Another series of studies involved treating the vessels with indomethacin for 10 min before precontacting the vessels with phenylephrine (10 Ϫ7 M).…”

Inhibition of Soybean Lipoxygenases – Structural and Activity Models for the Lipoxygenase Isoenzymes Family

“…It was pointed out by Walther et al, that a course of inhibition, by the drug ebselen, (noncompetitive vs competitive) and its reversibility depend on the oxidation state of iron, i.e. whether the enzyme is catalytically silent with Fe 2+ when it binds covalently, causing irreversible inhibition or preoxidized and active with Fe 3+ in the presence of the fatty acid substrate (Walther et al, 1999). In both cases the enzyme's performance can be illustrated by a classic Lineweaver-Burk plot.…”

Discovery of (2-Fluoro-Benzyl)-(2-Methyl-2-Phenethyl-2<i>H</i>-Chromen-6-yl)-Amine (KRH-102140) as an Orally Active 5-Lipoxygenase Inhibitor with Activity in Murine Inflammation Models

“…Enzyme assays of 12-LO and 15-LO were carried out in a similar manner to 5-LO, but devoid of ATP. Hinokitiol and ebselen were used as positive controls for 12-LO and 15-LO, respectively, as reported previously [14,15] .…”