Introduction: Lipoxygenases are widely distributed throughout the plant and animal kingdoms and play a central role in their biology. In plants they are involved in germination and senescence. In mammalian tissue, there are three major human lipoxygenases (HLO), 5-, 12-, and 15-HLO, whose primary enzymatic difference is their positional specificity on arachidonic acid (AA). These lipoxygenase products are the precursors of hormones such as leukotrienes and lipoxins which have been implicated as critical signaling molecules in a variety of inflammatory diseases and cancers. 5-HLO products cause asthma, while 12-HLO products play a major role in psoriasis and 15-HLO products may initiate the primary stage of atherosclerosis. In addition, these lipoxygenase isozymes are also involved in uncontrolled cell growth and/or regulation. 15-HLO has been shown to be a key biological agent in colorectal cancers, while 12-HLO is involved in pancreatic, breast and prostate cancers. 5-HLO is up-regulated in prostate cancer and its inhibition abolishes all cell proliferation, inducing apoptosis. These broad implications in human disease and cancer have elicited great interest in lipoxygenase as a potential therapeutic target and have motivated our lab to investigate lipoxygenase activity and inhibition.

Lipoxygenase Enzyme Mechanism: In order to develop lipoxygenase inhibitors, one needs to understand its enzymatic function. Our lab achieves this by studying three lipoxygenase enzymes, soybean 15-LO (SLO), human 12-LO (12-HLO) and human 15-LO (15-HLO) through a variety of spectroscopic and kinetic methods. The soybean enzyme is more robust than the human enzymes so it is used as a model system to develop a detailed mechanism for lipoxygenase as a class of enzyme. We have subsequently utilized this mechanism of SLO to compare with the human enzymes and determined that their mechanisms are remarkably similar despite their 100-fold difference in rate. We are now expanding this investigation by using time-resolved crystallography and stopped-flow spectroscopy to see mechanistic intermediates on the atomic level. These two techniques are complimentary in that the crystallography observes changes in the protein structure while the spectroscopic methods focus on the metal environment.

High Thru-Put Inhibitor Discovery: Our lab is also interested in the discovery and characterization of novel inhibitors to lipoxygenase. We currently have discovered over 20 unique lipoxygenase inhibitors through screening the marine natural products (MNP) library of our collaborator, Prof. Phil Crews. The library of Prof. Crews' is one of the worlds largest and offers a unique source of novel chemical structures unavailable through standard synthetic or combinatorial methods. We shall screen this library with a newly developed high thru-put lipoxygenase assay which should increase our inhibitor discovery dramatically. These inhibitors will then be investigated with biochemical and spectroscopic methods to determine how they bind and inhibit lipoxygenase.

Proteomic Investigations of Lipoxygenase: The final area of research in our lab is investigating the biological effect of these inhibitors on the overall biochemistry of the cancer cell with proteomics. Proteomics is the study of the induction and/or repression of all the proteins in a particular cell line. We are challenging prostrate cancer cells with our novel lipoxygenase inhibitors and determining the biological consequence of their addition. In this manner, we will determine not only the effectiveness of our inhibitors in killing the cancer cells but also how they affect the biochemical pathway of a cell. In this manner, we will classify our novel inhibitors by their potency and mode of action, which will help us design the next generation of lipoxygenase pharmaceuticals, that will target either the catalytic or allosteric sites   and be selective towards 12-HLO or 15-HLO.