Structural and Therapeutic Investigations of Human Lipoxygenase

The research in this dissertation describes the investigations of potential therapeutics as well as structural and allosteric properties of human lipoxygenases. Lipoxygenases (LOX) are a ubiquitous enzyme found in plants and mammals, of which are responsible for regulation of inflammation in humans. Uncontrolled inflammation in humans may result in various types of cancers and inflammatory diseases, for which LOX is implicated. This has prompted the Holman lab to explore a diverse range on potential therapeutic targets in hopes of discovery of novel selective LOX inhibitors, while concurrently investigating the structural and kinetic properties of the enzyme.Through use of conventional kinetic and structural studies we investigated the role of the polycystin-1 lipoxygenase alpha-toxin (PLAT) domain's role in enzyme catalysis and allosteric regulation. Previous studies had implicated the PLAT domain as being a critical aspect of the allosteric binding site. This theory was explored through extensive investigations into the resulting effects elicited by removal of the PLAT domain from human epithelial 15-lipoxygenase-2 (15-LOX-2). In chapter 2 we present our findings supporting our previous concept, that indeed the PLAT domain plays a key role in the allosteric properties of 15-LOX-2.Chapter 3 describes collaboration with the National Institutes Chemical Genomic Center, where we report the discovery of a novel dual inhibitor targeting fungal sterol 14á-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual selective inhibitor against human 5-LOX and CYP51 in vitro.Understanding the mode of action of lipoxygenase (LOX) inhibitors is critical to determining their efficacy in the cell. The pseudoperoxidase assay is an important tool for establishing if an inhibitor is reductive in nature. In chapter 4, we evaluate the effectiveness of two distinct pseudoperoxidase methods in characterizing known inhibitor's redox properties; the "234 nm" decomposition and xylenol orange assay. In addition, we identified rapid inactivation occurring with particular inhibitors in the pseudoperoxidase assay. To account for the resulting inaccuracy attributed to this inhibitor dependent inactivation, we modified the pre-existing "234 nm" assay allowing for observation of this inactivation