UC Santa Cruz

The research in this dissertation describes the discovery of potent and selective inhibitors of various human lipoxygenases (LOXs). The binding modes of specific inhibitors and substrates in the active site of human 15-lipoxygenase-1 (h15-LOX-1) were also investigated. Oxo-lipids, a large family of oxidized human lipoxygenase products, are involved in different inflammatory responses in the cell. Oxo-lipids contain electrophilic sites that can potentially form covalent bonds through a Michael addition mechanism with nucleophilic residues in protein active sites and 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 of all the oxo-lipids studied, with an IC50 = 1 ± 0.1 μM and was highly selective. Time-dependent studies did not demonstrate irreversible inhibition with 15-oxo-ETE, however, this does not preclude the role of its Michael acceptor in increasing its potency. 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. 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.

Epithelial h15-LOX-2 is of clinical interest due to its link in the progression of macrophages to foam cells, which are present in atherosclerotic plaques. Coronary artery disease is the primary cause of deaths in men and women in America. The discovery of novel inhibitors that are selective and potent against h15-LOX-2 may aid in identifying this protein’s role in heart disease. Two novel molecular scaffolds, (MLS000545091 and MLS000536924), were identified using High Throughput Screening (HTS) that are selective and potent against h15-LOX-2.

Human reticulocyte 15-Lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h15-LOX-1 is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood-brain barrier leakage. Stroke is the fifth leading cause of death and the first leading cause of disability in America. The discovery of inhibitors that selectively target recombinant h15-LOX-1 in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. A new family of inhibitors was identified in a High Throughput Screen that are selective and potent against recombinant h15-LOX-1 and cellular mouse 15-LOX (m15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC50 potency of 3.4 ± 0.5 μM against h15-LOX-1 in vitro and an ex vivo IC50 potency of approximately 10 μM in a mouse neuronal cell line, HT-22. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h15-LOX-1 in vitro and m15-LOX ex vivo.

The discovery of h15-LOX-1 inhibitors could potentially be novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction due to the lack of a protein crystal structure. Site-directed mutagenesis and molecular modeling were utilized to gain a better structural understanding of inhibitor interactions with the active site of h15-LOX-1. Eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 were generated to determine whether these active site residues interact with two structurally similar h15-LOX-1 inhibitors, a ML094 derivative and ML351. IC50 values and steady-state inhibition kinetics were determined with the eight mutants and four of the mutants affected inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that the ML094 derivative and ML351 bind to similar sites in the active site but have subtle differences in their binding modes.

Lipoxygenases are capable of catalyzing dioxygenation and dehydration reactions when reacting with conjugated mono-hydroperoxy fatty acids. Dioxygenation of mono-hydroperoxide fatty acids lead to di-hydroperoxide products while dehydration of mono-hydroperoxides followed by hydrolysis lead to di-hydroxide products. The fatty acid kinetics, product profile and mechanism of product formation was investigated between WT human 15-lipoxygenase-1 and 15S-HpETE in comparison to the reaction between an active site mutant, F414I, and 15S-HpETE. The substrate affinity and kcat of 15S-HpETE turnover were comparable for both WT h15-LOX-1 and F414I. Alternatively, the substrate affinity and kcat of AA turnover varied for WT h15-LOX-1 and F414I leading to a decreased catalytic efficiency of AA turnover by F414I relative to WT h15-LOX-1. The kinetic data suggests that the active site mutant, F414I, is less efficient at turning over AA relative to WT but comparable at turning over 15S-HpETE. 5S,15S-DiHpETE, 8S,15S-DiHETE and 14R,15S-DiHpETE were identified as the major products in the reaction for both WT 15-LOX-1 and F414I, possibly indicating that this active site mutation does not significantly affect the binding mode of 15S-HpETE in the active site. Comparison of the mass spectra of the reduced and unreduced reactions of 15S-HpETE and both enzymes reveal that 14R,15S-DiHpETE and 5S,15S-DiHETE are dioxygenation products while 8S,15S-DiHETE is the hydrolyzed dehydration product. In contrast to rabbit 15-LOX, h15-LOX-1 does not react with 15S-DiHETE with or without 13S-HpODE.