Inhibitory and mechanistic investigations of oxo-lipids with human lipoxygenase isozymes

• Published in: Bioorganic & medicinal chemistry Vol. 22; no. 15; pp. 4293 - 4297
• Main Authors: Armstrong, Michelle M., Diaz, Giovanni, Kenyon, Victor, Holman, Theodore R.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.08.2014
• Subjects: Animals; Arachidonic Acids - chemistry; Arachidonic Acids - metabolism; Glycine max - enzymology; Humans; Inhibitor; Isoenzymes - antagonists & inhibitors; Isoenzymes - metabolism; Kinetics; Lipoxygenase; Lipoxygenase Inhibitors - chemistry; Lipoxygenase Inhibitors - metabolism; Lipoxygenases - chemistry; Lipoxygenases - metabolism; Mechanism; Oxo-lipids; Protein Binding; Rabbits; Substrate Specificity; s15-LOX-1; fmk; 12-oxo-ETEre; h12-LOX; RSK1/2; Oxo-lipids; h5-LOX; Mechanism; 13-HpODE; 12-HEDH; 5-HEDH; r15-LOX; 15-HpETE; GSH; AA; CF; 12-HETE; 15-PGDH; IEC; 12-HpETE; 13-HODE; 5-HpETE; 12-HETrE; 13-oxo-ODE; PPARγ; 12-oxo-ETE; LOX; LA; h15-LOX-2; CYP2S1; h15-LOX-1; Inhibitor; Lipoxygenase; hLOX; 5-oxo-ETE; 15-oxo-ETE
• ISSN: 0968-0896; 1464-3391
• DOI: 10.1016/j.bmc.2014.05.025

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±0.1μM 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±0.008μM and a Kiu value of 2.10±0.8μM. 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±13.2μM) and the time frame (k2=0.0019±0.00032s−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.