title: The environment of the lipoxygenase iron binding site explored with novel hydroxypyridinone iron chelators.
abstract: The mechanisms of lipoxygenase inhibition by iron chelators have been investigated in human neutrophils and in isolated soybean lipoxygenase. Their Fe(III)-containing active sites have been targeted by synthesizing novel bidentate chelators from the hydroxypyridinone family sufficiently small to gain access through the hydrophobic channels of lipoxygenase. In stimulated human neutrophils, release of [3H]arachidonate-labeled eicosanoids is dependent on the lipid solubility of hydroxypyridinones, but larger hexadentate chelators have no effect on this or on total cellular leukotriene B4 production. Lipophilic hydroxypyridinones inhibit 5-lipoxygenase at equivalent concentrations to the established inhibitor, piriprost, and show additional but minor anti-phospholipase A2 activity. Soybean 15-lipoxygenase inhibition is also dependent on the lipid solubility and coordination structure of chelators. Inhibition is associated with the formation of chelate-iron complexes, which are removed by dialysis without restoration of enzyme activity. Only after adding back iron is activity restored. Electron paramagnetic resonance studies show the removal of the iron center signal (g = 6) is concomitant with formation of Fe(III)-chelator complexes, identical in spectral shape and g value to 3:1 hydroxypyridinone Fe(III) complexes. Removal of iron is not the only mechanism by which hydroxypyridinones can inhibit lipoxygenase in intact cells, however, as a lipophilic non-iron-binding hydroxypyridinone, which shows no inhibition of the soybean lipoxygenase activity, partially inhibits 5-lipoxygenase in intact neutrophils without inhibiting neutrophil phospholipase A2.
authors.affiliation: Department of Clinical Hematology, University College London Medical School, WC1E 6HX London, United Kingdom.
authors.forename: R D
authors.initials: RD
authors.lastname: Abeysinghe
authors.forename: P J
authors.initials: PJ
authors.lastname: Roberts
authors.forename: C E
authors.initials: CE
authors.lastname: Cooper
authors.forename: K H
authors.initials: KH
authors.lastname: MacLean
authors.forename: R C
authors.initials: RC
authors.lastname: Hider
authors.forename: J B
authors.initials: JB
authors.lastname: Porter
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007501:Iron / Q000737:chemistry; D007502:Iron Chelating Agents / Q000737:chemistry; D008024:Ligands; D008084:Lipoxygenase / Q000737:chemistry*; D016859:Lipoxygenase Inhibitors / Q000737:chemistry; D009504:Neutrophils / Q000201:enzymology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011728:Pyridones / Q000737:chemistry; D012995:Solubility; D013025:Glycine max / Q000201:enzymology; D013481:Superoxides / Q000378:metabolism
chemical_list: D007502:Iron Chelating Agents; D008024:Ligands; D016859:Lipoxygenase Inhibitors; D011728:Pyridones; D013481:Superoxides; D007501:Iron; D008084:Lipoxygenase; D010741:Phospholipases A; D054467:Phospholipases A2