Pseudoperoxidase activity of 5-lipoxygenase stimulated by potent benzofuranol and N-hydroxyurea inhibitors of the lipoxygenase reaction

Abstract

The purified 5-lipoxygenase from porcine leukocytes was found to catalyse the degradation of lipid hydroperoxides in the presence of potent inhibitors of the lipoxygenase reaction. Derivatives of diphenyl-N-hydroxyureas, 4-hydroxybenzofurans and 5-hydroxydihydrobenzofurans all stimulated the 5-lipoxygenase-mediated destruction of 13-hydroperoxyoctadecadienoic acid (13-HPOD). The reaction was dependent on inhibitor and hydroperoxide concentrations (1-10 microM) and could not be detected using heat-inactivated enzyme, when ATP and Ca2+ were omitted or when the hydroperoxide was replaced by the corresponding alcohol. The stability of the inhibitors during this pseudoperoxidase reaction was investigated by measuring the recoveries of 5-hydroxy-2-phenethyl-6-(3-phenoxypropyl)-2,3-dihydrobenzofuran and N-(4-chlorophenyl)-N-hydroxy-N'-(3-chlorophenyl)urea from the reaction mixtures using reverse-phase h.p.l.c. By using an equimolar concentration of 13-HPOD and inhibitor (10 microM) and under conditions where 50% of the 13-HPOD was consumed, the concentration of the benzofuranol decreased by 30%, whereas the N-hydroxyurea derivative could be completely recovered from the reaction mixture. A stimulation of the pseudoperoxidase reaction could be detected only with very effective inhibitors of leukotriene B4 biosynthesis by human leucocytes [IC50 (concn. causing 50% inhibition) less than 100 nM], but not with closely related structural analogues of lower potency or other inhibitors such as nordihydroguaiaretic acid, quercetin or the hydroxamate A-64077. These results demonstrate that 5-lipoxygenase possesses a pseudoperoxidase activity and indicate that potent inhibitors in both N-hydroxyurea and benzofuranol series can function as reducing agents for the enzyme.

Baicalein is a potent in vitro inhibitor against both reticulocyte 15-human and platelet 12-human lipoxygenases

“…Pseudoperoxidase studies of 15-hLO-1 were performed as previously described to determine if a particular inhibitor could function as a reductant to the active site iron. 47 Baicalein's mode of inhibition against 15-hLO-1 proved to follow a redox mechanism as seen previously with NDGA and other catechol like compounds, 19 while apigenin followed reversible binding inhibition. It should be noted that the pseudoperoxidase activity was reliable and consistent with 15-hLO-1, but for 12-hLO, only the inhibitor BWB70C could support the assay.…”

“…Numerous other catechol inhibitors are reductive inhibitors 19 but it had never been directly demonstrated whether baicalein was a reductive inhibitor against human LO or not. Utilizing the established psuedoperoxidase assay 47 , we show that baicalein is a reductive inhibitor against 15-hLO-1. Based on our knowledge of baicalein, it is reasonable to assume that a catecholic alcohol ligates the iron and causes an inner sphere reduction on the active site iron, with baicalein undergoing oxidation to its quinone form ( Figure 5).…”

“…Pseudoperoxidase activity of both 12-hLO and 15-hLO-1 was determined as previously described. 47 Pseudoperoxidase activity was monitored by following the degradation of 13(S)-hydroperoxyoctadecadieneoic acid (13-HPOD) at 234 nm. All reactions were performed in 2mL of buffer at room temperature (23°C), with a known lipoxygenase redox inhibitor BWB70C as the control.…”

Dimerization of human 5-lipoxygenase

“…Alternatively, for COX which catalyzes the initial steps in prostaglandin biosynthesis by the oxygenation of arachidonic acid, Yuan et al (2009) showed that COX forms dimers, where one substrate molecule binds with high affi nity to one COX site and facilitates the oxygenation of arachidonic acid by the other catalytic partner (Yuan et al , 2009 ). According to these data we assumed that one monomer of the 5-LO could be the catalytically active partner whereas the other monomer has a regulatory function, e.g., via its pseudoperoxidase activity (Riendeau et al , 1991 ). By gel fi ltration, native PAGE and LILBID-MS analysis, we found that 5-LO can form dimers and that dimer formation is enhanced in the presence of T20 micelles.…”

Attenuation of Cataract Progression by A-3922, a Dihydrobenzofuran Derivative, in Streptozotocin-Induced Diabetic Rats

“…23) A recent report indicated that a dihydrobenzofuran derivative A-3922 effectively inhibited corneal neovascularization induced by linoleic acid hydroperoxide in rabbits. 24) Here, we examined the effect of A-3922 on the cataract formation in streptozotocin-induced (STZ-induced) diabetic rats in vivo.…”