Investigating The Inhibition Mechanism Of Lipoxygenases By Oxo-lipids

Human lipoxygenases hydroperoxidate specific carbons of arachidonic acid to initiate the production of leukotrienes and lipoxins involved in asthmatic reactions and inflammatory diseases, respecively. Lipoxygenases biological implications in disease make them ideal targets for pharmaceutical intervention. Oxo-lipid production is initiated by lipoxygenase and has been implicated in a variety of diseases but the oxo-lipids’ biological roles in the cell remains to be elucidated. 5-oxoETE has shown to be involved in the asthmatic inflammatory response through the stimulation of neutrophil and eosinophil migration to the airways. 13-oxoODE has demonstrated anti-inflammatory effects through interactions with peroxisome proliferator-activated receptor gamma. 15-oxoETE has been found in mast cells and 12-oxoETE levels are elevated during psoriasis, however their respective roles in the cell remain unknown. Currently, no studies have been performed to characterize the inhibitory interactions between oxo-lipids and lipoxygenases. Such studies could prove to be useful in deciphering the biological implications of 12oxoETE and 15oxoETE. The synthesis of 5-oxoETE, 12-oxoETE, 15-oxoETE and 13-oxoODE were performed via a two-step reaction and purified by High Performance Liquid Chromatography. Comprehensive studies were conducted to determine the potency of these oxo-lipids against our lipoxygenase isozymes, 5-LOX, 12-LOX, 15-LOX-1, 15-LOX-2, rabbit LOX and soybean LOX. These studies have concluded that oxo-lipids selectively inhibit lipoxygenases at levels that may be biologically relevant. The Michael addition reaction was hypothesized to be the mechanism of inhibition, however incubation studies yielded no significant time dependence and warrants future experiments. We will be exploring the type of inhibition through use of steady-state inhibition kinetics.