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Characterization and Inhibition Studies of 15-Lipoxygenase Enzymes

  • Author(s): Jameson, John Brian
  • Advisor(s): Holman, Theodore R
  • et al.
Abstract

The research presented in this dissertation focuses on two distinct areas of 15-lipoxygenase biology: screening and identification of inhibitors toward human 15-lipoxygenase enzymes and biological and biochemical characterization of a 15-lipoxygenase enzyme (p15-LOX) from the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). Two novel, selective inhibitors are reported for human 15-lipoxygenase-2 (15-LOX-2). The reported compounds were micromolar active with competitive and mixed type inhibition. They will serve as chemical tools for future studies to more deeply probe the role of 15-LOX-2 in atherosclerosis, cancer, and for general elucidation of 15-LOX-2 function in tissues. In addition, the binding affinities for five nanomolar active human 15-lipoxygenase-1 inhibitors were determined by a novel mass spectrometric assay with the ability to measure binding affinities well below the current limit of quantitation for the traditional UV assay. The assay also represents the first of its kind capable of assessing allosteric effect in a high throughput, 96-well format, opening the possibility of screening compound libraries to identify allosteric effectors to lipoxygenase (LOX) enzymes.

Finally, a number of biochemical and biological properties of the LOX enzyme expressed by P. aeruginosa were determined. The most actively metabolized substrate is AA and Kcat far exceeds human homologues at low pH. The enzyme is active with a variety of polyunsaturated fatty acid substrates, but phospholipids are not metabolized well compared to human LOX enzymes. Selective inhibitors to human LOX isozymes do not significantly inhibit p15-LOX. Expression of LOX during P. aeruginosa infection increases observed cytokines from several cell types. Together these studies support the hypothesis that this enzyme is capable of disrupting host immunity, but do not strongly support expression of p15-LOX solely for this purpose. Subsequent studies are needed to further elucidate the biological function of p15-LOX.

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