UC Santa Cruz

"Abstract"

"ALLOSTERIC AND INHIBITORY INVESTIGATIONS OF 15- HUMAN LIPOXYGENASES"

by

Netra S. Joshi

This dissertation focuses on two isoforms of 15-human lipoxygenase, reticulocyte 15-LOX-1 and epithelial 15-LOX-2, that differ considerably in their tissue expression, reaction specificity, auto-inactivation and response to the allosteric effectors. These 15-LOX isoforms exert their function in the cells by reacting with a variety of endogenous fatty acid substrates and producing hydroperoxy products, which regulate the inflammatory process in the cells. These 15-LOX products can participate in opposing roles in human diseases, and their ratios are known to regulate cell-cell adhesion processes in cells, playing a vital role in inflammation, cancer and thrombosis. Therefore, understanding the factors that affect the substrate specificity of these two LOX isozymes is critical to comprehend their exact role in human diseases. Furthermore, 15-LOX-1 has been recently implicated in stroke related damage via hydroperoxidation of fatty acid containing membranes and its ability to degrade mitochondrial membrane when triggered by reactive oxygen species. Studies indicate the potential of 15-LOX-1 inhibitors as neuroprotectors against stroke related damage. However drug discovery for stroke therapeutics is a long, tedious process with high chance of failure and presents a constant search for potent, selective inhibitors

This dissertation investigates the discovery of a novel chemotype for 15-LOX-1 inhibitors. It describes in detail the initial screening, characterization and optimization of potent and selective inhibitors of 15-LOX-1. The results of the in-vivo and in-vitro studies demonstrate the potential of this chemotype as a stroke therapeutic. Finally, the factors that regulate the substrate specificity of human epithelial 15-LOX-2 are also investigated here. The findings demonstrate that both pH and LOX products alter the kinetic parameters of C20 and C18 fatty acid substrates differentially, with both pH and LOX products activating the C20 kinetics but both inhibit C18 kinetics, resulting into a significant increase in the C20/C18 substrate specificity ratio. These alterations in the product ratio may present important consequences in human disease progression due to the distinct biological effects of these products.