Inhibition of human leukocyte 5-lipoxygenase by a 4-hydroxybenzofuran, L-656,224. Evidence for enzyme reduction and inhibitor degradation.
Detailed studies of the interaction of L-656,224 (2-[(4'-methoxyphenyl)methyl]-3-methyl-4-hydroxy-5-propyl-7- chlorobenzofuran) with 5-lipoxygenase were conducted using the enzymes from human and pig leukocytes. L-656,224 was a potent inhibitor of these 5-lipoxygenases although its efficiency varied with enzyme concentration. L-656,224 also stimulated the pseudoperoxidase activity of 5-lipoxygenase as measured by the consumption of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD), indicating that this compound can reduce the enzyme. Furthermore the inhibitor was degraded rapidly by both cell-free leukocyte extracts and purified 5-lipoxygenase after incubation with 13-HPOD, ATP and calcium ions. The degradation of L-656,224 was also observed during inhibition of the lipoxygenase reaction and occurred mainly after the initial lag phase of the reaction when hydroperoxides begin to accumulate. A single major radioactive product was formed after incubation of [3H]L-656,224 with purified 5-lipoxygenase in the presence of 13-HPOD. This product was unstable and could not be isolated. During the course of the pseudoperoxidase reaction, [3H]L-656,224 covalently labelled the enzyme, suggesting that a chemically reactive species had been formed. These data are consistent with the hypothesis that L-656,224 reduces the oxidized form of the 5-lipoxygenase to an inactive form, with degradation of the inhibitor and regeneration of the active enzyme with hydroperoxides.
Rouzer
C A
CA
Department of Pharmacology, Merck Frosst Centre for Therapeutic Research, Québec, Canada.
Riendeau
D
D
Falgueyret
J P
JP
Lau
C K
CK
Gresser
M J
MJ
D000255:Adenosine Triphosphate; D000478:Alkylation; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D001572:Benzofurans / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium; D006801:Humans; D007962:Leukocytes / Q000187:drug effects* / Q000201:enzymology; D008041:Linoleic Acids / Q000378:metabolism; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism
D001572:Benzofurans; D008041:Linoleic Acids; D008054:Lipid Peroxides; D016859:Lipoxygenase Inhibitors; C055868:L 656224; C038649:13-hydroperoxy-9,11-octadecadienoic acid; D000255:Adenosine Triphosphate; D010544:Peroxidases; D001094:Arachidonate 5-Lipoxygenase; D002118:Calcium