Inhibitory and mechanistic investigations of oxo-lipids with human lipoxygenase isozymes |
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| Affiliation: | 1. Chemical Biology Laboratory, National Cancer Institute, 376 Boyles St., Frederick, MD 21702, USA;2. Basic Science Program, Chemical Biology Laboratory, Leidos Biomedical Research, Inc., National Cancer Institute, 376 Boyles St., Frederick, MD 21702, USA;1. Department of Organic Chemistry, University of Debrecen, POB 20, H-4010 Debrecen, Hungary;2. Department of Inorganic and Analytical Chemistry, University of Debrecen, POB 21, H-4010 Debrecen, Hungary;3. Department of Medical Chemistry, Medical and Health Science Centre, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary;4. Institute of Biology, Medicinal Chemistry and Biotechnology, The National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, GR-116 35 Athens, Greece;1. Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, United States;2. Cardiovascular Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, United States;3. The Bert W. Strassburger Lipid Center, Tel Aviv University, 52621 Hashomer Tel, Israel;4. Sheba Medical Center and Sackler School of Medicine, Tel Aviv University, 52621 Hashomer Tel, Israel;5. Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390, United States |
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| Abstract: | Oxo-lipids, a large family of oxidized human lipoxygenase (hLOX) products, are of increasing interest to researchers due to their involvement in different inflammatory responses in the cell. Oxo-lipids are unique because they contain electrophilic sites that can potentially form covalent bonds through a Michael addition mechanism with nucleophilic residues in protein active sites and thus increase inhibitor potency. Due to the resemblance of oxo-lipids to LOX substrates, the inhibitor potency of 4 different oxo-lipids; 5-oxo-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5-oxo-ETE), 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE), 12-oxo-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid (12-oxo-ETE), and 13-oxo-9,11-(Z,E)-octadecadienoic acid (13-oxo-ODE) were determined against a library of LOX isozymes; leukocyte 5-lipoxygenase (h5-LOX), human reticulocyte 15-lipoxygenase-1 (h15-LOX-1), human platelet 12-lipoxygenase (h12-LOX), human epithelial 15-lipoxygenase-2 (h15-LOX-2), soybean 15-lipoxygenase-1 (s15-LOX-1), and rabbit reticulocyte 15-LOX (r15-LOX). 15-Oxo-ETE exhibited the highest potency against h12-LOX, with an IC50 = 1 ± 0.1 μM and was highly selective. Steady state inhibition kinetic experiments determined 15-oxo-ETE to be a mixed inhibitor against h12-LOX, with a Kic value of 0.087 ± 0.008 μM and a Kiu value of 2.10 ± 0.8 μM. Time-dependent studies demonstrated irreversible inhibition with 12-oxo-ETE and h15-LOX-1, however, the concentration of 12-oxo-ETE required (Ki = 36.8 ± 13.2 μM) and the time frame (k2 = 0.0019 ± 0.00032 s−1) were not biologically relevant. These data are the first observations that oxo-lipids can inhibit LOX isozymes and may be another mechanism in which LOX products regulate LOX activity. |
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| Keywords: | Lipoxygenase Oxo-lipids Inhibitor Mechanism |
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