Kinetic, Mechanistic, and Structural Investigations of Human Lipoxygenases

The research contained in this dissertation examines the kinetic, mechanistic, and structural basis of human lipoxygenase (LOX) catalysis. LOX are found throughout the plant and animals kingdoms, and in humans LOX are involved in inflammatory signaling. Through this involvement in the inflammatory process and the ability to oxidize membrane bound polyunsaturated fatty acids (PUFA), LOX have been implicated in a variety of cancers and disease states. The impact of substrate saturation on platelet 12-LOX kinetics and mechanism was investigated in Chapter 2. The platelet signaling pathways and implications on platelet aggregation of these substrates were determined. Additionally, the novel allosteric effect of 15S-HpEPE on 12-LOX mechanism was probed. Chapter 3 scrutinized a previously discovered lipoxygenase activator. This dissertation works shows that the activator is LOX isoform specific, with only h15-LOX-1 being kinetically activated in the presence of the compound. Furthermore, the activator was found to activate the catalysis of shorter PUFA substrates, AA and LA, but not that of the longer SPM precursor, DHA. The implications of this finding has consequences on the potential therapeutic development of the activating compound. The effect on enzyme kinetics and mechanism was assayed, and a V-type activation was observed. Chapter 4 builds off of a previous discovery in our lab that demonstrated the altered positional specificity of h15-LOX-1 when reacting with the metabolites of 5-LOX. In this chapter, site-directed mutagenesis was performed on key active site residues, with the kinetic and mechanistic implications of those residues determined experimentally. Additionally, the consequence of the altered positional specificity with regards to the biosynthesis of the potent SPM, Resolvin E4, was examined

Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors

Structural basis for altered positional specificity of 15-lipoxygenase-1 with 5S-HETE and 7S-HDHA and the implications for the biosynthesis of resolvin E4

Human 15-lipoxygenases (LOX) are critical enzymes in the inflammatory process, producing various pro-resolution molecules, such as lipoxins and resolvins, but the exact role each of the two 15-LOXs in these biosynthetic pathways remains elusive. Previously, it was observed that h15-LOX-1 reacted with 5S-HETE in a non-canonical manner, producing primarily the 5S,12S-diHETE product. To determine the active site constraints of h15-LOX-1 in achieving this reactivity, amino acids involved in the fatty acid binding were investigated. It was observed that R402L did not have a large effect on 5S-HETE catalysis, but F414 appeared to π-π stack with 5S-HETE, as seen with AA binding, indicating an aromatic interaction between a double bond of 5S-HETE and F414. Decreasing the size of F352 and I417 shifted oxygenation of 5S-HETE to C12, while increasing the size of these residues reversed the positional specificity of 5S-HETE to C15. Mutants at these locations demonstrated a similar effect with 7S-HDHA as the substrate, indicating that the depth of the active site regulates product specificity for both substrates. Together, these data indicate that of the three regions proposed to control positional specificity, π-π stacking and active site cavity depth are the primary determinants of positional specificity with 5S-HETE and h15-LOX-1. Finally, the altered reactivity of h15-LOX-1 was also observed with 5S-HEPE, producing 5S,12S-diHEPE instead of 5S,15S-diHEPE (aka resolvin E4 (RvE4). However, h15-LOX-2 efficiently produces 5S,15S-diHEPE from 5S-HEPE. This result is important with respect to the biosynthesis of the RvE4 since it obscures which LOX isozyme is involved in its biosynthesis. Future work detailing the expression levels of the lipoxygenase isoforms in immune cells and selective inhibition during the inflammatory response will be required for a comprehensive understanding of RvE4 biosynthesis

Reevaluation of a Bicyclic Pyrazoline as a Selective 15-Lipoxygenase V‑Type Activator Possessing Fatty Acid Specificity

Regulation of lipoxygenase (LOX) activity is of great interest due to the involvement of the various LOX isoforms in the inflammatory process and hence many diseases. The bulk of investigations have centered around the discovery and design of inhibitors. However, the emerging understanding of the role of h15-LOX-1 in the resolution of inflammation provides a rationale for the development of activators as well. Bicyclic pyrazolines are known bioactive molecules that have been shown to display antibiotic and anti-inflammatory activities. In the current work, we reevaluated a previously discovered bicyclic pyrazoline h15-LOX-1 activator, PKUMDL_MH_1001 (written as 1 for this publication), and determined that it is inactive against other human LOX isozymes, h5-LOX, h12-LOX, and h15-LOX-2. Analytical characterization of 1 obtained in the final synthesis step identified it as a mixture of cis- and trans-diastereomers: cis-1 (12%) and trans-1 (88%); and kinetic analysis indicated similar potency between the two. Using compound 1 as the cis-trans mixture, h15-LOX-1 catalysis with arachidonic acid (AA) (AC50 = 7.8 +/- 1 μM, A max = 240%) and linoleic acid (AC50 = 5.3 +/- 0.7 μM, A max = 98%) was activated, but not with docosahexaenoic acid (DHA) or mono-oxylipins. Steady-state kinetics demonstrate V-type activation for 1, with a β value of 2.2 +/- 0.4 and an K x of 16 +/- 1 μM. Finally, it is demonstrated that the mechanism of activation for 1 is likely not due to decreasing substrate inhibition, as was postulated previously. 1 also did not affect the activity of the h15-LOX-1 selective inhibitor, ML351, nor did 1 affect the activity of allosteric effectors, such as 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) and 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-HpDHA). These data confirm that 1 binds to a distinct activation binding site, as previously postulated. Future work should be aimed at the development of selective activators that are capable of activating h15-LOX-1 catalysis with DHA, thus enhancing the production of DHA-derived pro-resolution biomolecules

DHA 12- LOX- derived oxylipins regulate platelet activation and thrombus formation through a PKA- dependent signaling pathway

BackgroundThe effects of docosahexaenoic acid (DHA) on cardiovascular disease are controversial and a mechanistic understanding of how this omega- 3 polyunsaturated fatty acid (Ï - 3 PUFA) regulates platelet reactivity and the subsequent risk of a thrombotic event is warranted. In platelets, DHA is oxidized by 12- lipoxygenase (12- LOX) producing the oxidized lipids (oxylipins) 11- HDHA and 14- HDHA. We hypothesized that 12- LOX DHA- oxylipins may be involved in the beneficial effects observed in dietary supplemental treatment with Ï - 3 PUFAs or DHA itself.ObjectivesTo determine the effects of DHA, 11- HDHA, and 14- HDHA on platelet function and thrombus formation, and to elucidate the mechanism by which these Ï - 3 PUFAs regulate platelet activation.Methods and resultsDHA, 11- HDHA, and 14- HDHA attenuated collagen- induced human platelet aggregation, but only the oxylipins inhibited - ºIIbβ3 activation and decreased - º- granule secretion. Furthermore, treatment of whole blood with DHA and its oxylipins impaired platelet adhesion and accumulation to a collagen- coated surface. Interestingly, thrombus formation was only diminished in mice treated with 11- HDHA or 14- HDHA, and mouse platelet activation was inhibited following acute treatment with these oxylipins or chronic treatment with DHA, suggesting that under physiologic conditions, the effects of DHA are mediated through its oxylipins. Finally, the protective mechanism of DHA oxylipins was shown to be mediated via activation of protein kinase A.ConclusionsThis study provides the first mechanistic evidence of how DHA and its 12- LOX oxylipins inhibit platelet activity and thrombus formation. These findings support the beneficial effects of DHA as therapeutic intervention in atherothrombotic diseases.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167023/1/jth15184-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167023/2/jth15184.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167023/3/jth15184_am.pd

Fatty acids negatively regulate platelet function through formation of noncanonical 15‐lipoxygenase‐derived eicosanoids

The antiplatelet effect of polyunsaturated fatty acids is primarily attributed to its metabolism to bioactive metabolites by oxygenases, such as lipoxygenases (LOX). Platelets have demonstrated the ability to generate 15-LOX-derived metabolites (15-oxylipins); however, whether 15-LOX is in the platelet or is required for the formation of 15-oxylipins remains unclear. This study seeks to elucidate whether 15-LOX is required for the formation of 15-oxylipins in the platelet and determine their mechanistic effects on platelet reactivity. In this study, 15-HETrE, 15-HETE, and 15-HEPE attenuated collagen-induced platelet aggregation, and 15-HETrE inhibited platelet aggregation induced by different agonists. The observed anti-aggregatory effect was due to the inhibition of intracellular signaling including αIIbβ3 and protein kinase C activities, calcium mobilization, and granule secretion. While 15-HETrE inhibited platelets partially through activation of peroxisome proliferator-activated receptor β (PPARβ), 15-HETE also inhibited platelets partially through activation of PPARα. 15-HETrE, 15-HETE, or 15-HEPE inhibited 12-LOX in vitro, with arachidonic acid as the substrate. Additionally, a 15-oxylipin-dependent attenuation of 12-HETE level was observed in platelets following ex vivo treatment with 15-HETrE, 15-HETE, or 15-HEPE. Platelets treated with DGLA formed 15-HETrE and collagen-induced platelet aggregation was attenuated only in the presence of ML355 or aspirin, but not in the presence of 15-LOX-1 or 15-LOX-2 inhibitors. Expression of 15-LOX-1, but not 15-LOX-2, was decreased in leukocyte-depleted platelets compared to non-depleted platelets. Taken together, these findings suggest that 15-oxylipins regulate platelet reactivity; however, platelet expression of 15-LOX-1 is low, suggesting that 15-oxylipins may be formed in the platelet through a 15-LOX-independent pathway