Publications by authors named "Theodore R Holman"

80 Publications

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

J Thromb Haemost 2021 Mar 16;19(3):839-851. Epub 2020 Dec 16.

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA.

Background: The effects of docosahexaenoic acid (DHA) on cardiovascular disease are controversial and a mechanistic understanding of how this omega-3 polyunsaturated fatty acid (ω-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 ω-3 PUFAs or DHA itself.

Objectives: To determine the effects of DHA, 11-HDHA, and 14-HDHA on platelet function and thrombus formation, and to elucidate the mechanism by which these ω-3 PUFAs regulate platelet activation.

Methods And Results: DHA, 11-HDHA, and 14-HDHA attenuated collagen-induced human platelet aggregation, but only the oxylipins inhibited ⍺IIbβ3 activation and decreased ⍺-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.

Conclusions: This 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.
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http://dx.doi.org/10.1111/jth.15184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925359PMC
March 2021

Resolving the paradox of ferroptotic cell death: Ferrostatin-1 binds to 15LOX/PEBP1 complex, suppresses generation of peroxidized ETE-PE, and protects against ferroptosis.

Redox Biol 2021 Jan 16;38:101744. Epub 2020 Oct 16.

Children's Neuroscience Institute, University of Pittsburgh, Pittsburgh, PA, USA; Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA; Navigational Redox Lipidomics Group, Institute for Regenerative Medicine, IM Sechenov First Moscow State Medical University, Russian Federation. Electronic address:

Hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) is a ferroptotic cell death signal. HpETE-PE is produced by the 15-Lipoxygenase (15LOX)/Phosphatidylethanolamine Binding Protein-1 (PEBP1) complex or via an Fe-catalyzed non-enzymatic radical reaction. Ferrostatin-1 (Fer-1), a common ferroptosis inhibitor, is a lipophilic radical scavenger but a poor 15LOX inhibitor arguing against 15LOX having a role in ferroptosis. In the current work, we demonstrate that Fer-1 does not affect 15LOX alone, however, it effectively inhibits HpETE-PE production by the 15LOX/PEBP1 complex. Computational molecular modeling shows that Fer-1 binds to the 15LOX/PEBP1 complex at three sites and could disrupt the catalytically required allosteric motions of the 15LOX/PEBP1 complex. Using nine ferroptosis cell/tissue models, we show that HpETE-PE is produced by the 15LOX/PEBP1 complex and resolve the long-existing Fer-1 anti-ferroptotic paradox.
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http://dx.doi.org/10.1016/j.redox.2020.101744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596334PMC
January 2021

Role of Human 15-Lipoxygenase-2 in the Biosynthesis of the Lipoxin Intermediate, 5S,15S-diHpETE, Implicated with the Altered Positional Specificity of Human 15-Lipoxygenase-1.

Biochemistry 2020 10 13;59(42):4118-4130. Epub 2020 Oct 13.

Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California 95064, United States.

The oxylipins, 5S,12S-dihydroxy-6E,8Z,10E,14Z-eicosatetraenoic acid (5S,12S-diHETE) and 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5S,15S-diHETE), have been identified in cell exudates and have chemotactic activity toward eosinophils and neutrophils. Their biosynthesis has been proposed to occur by sequential oxidations of arachidonic acid (AA) by lipoxygenase enzymes, specifically through oxidation of AA by h5-LOX followed by h12-LOX, h15-LOX-1, or h15-LOX-2. In this work, h15-LOX-1 demonstrates altered positional specificity when reacting with 5S-HETE, producing 90% 5S,12S-diHETE, instead of 5S,15S-diHETE, with kinetics 5-fold greater than that of h12-LOX. This is consistent with previous work in which h15-LOX-1 reacts with 7S-HDHA, producing the noncanonical, DHA-derived, specialized pro-resolving mediator, 7S,14S-diHDHA. It is also determined that oxygenation of 5S-HETE by h15-LOX-2 produces 5S,15S-diHETE and its biosynthetic / flux is 2-fold greater than that of h15-LOX-1, suggesting that h15-LOX-2 may have a greater role in lipoxin biosynthesis than previously thought. In addition, it is shown that oxygenation of 12S-HETE and 15S-HETE by h5-LOX is kinetically slow, suggesting that the first step in the biosynthesis of both 5S,12S-diHETE and 5S,15S-diHETE is the production of 5S-HETE.
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http://dx.doi.org/10.1021/acs.biochem.0c00622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721368PMC
October 2020

Omega-6 DPA and its 12-lipoxygenase-oxidized lipids regulate platelet reactivity in a nongenomic PPARα-dependent manner.

Blood Adv 2020 09;4(18):4522-4537

Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI.

Arterial thrombosis is the underlying cause for a number of cardiovascular-related events. Although dietary supplementation that includes polyunsaturated fatty acids (PUFAs) has been proposed to elicit cardiovascular protection, a mechanism for antithrombotic protection has not been well established. The current study sought to investigate whether an omega-6 essential fatty acid, docosapentaenoic acid (DPAn-6), and its oxidized lipid metabolites (oxylipins) provide direct cardiovascular protection through inhibition of platelet reactivity. Human and mouse blood and isolated platelets were treated with DPAn-6 and its 12-lipoxygenase (12-LOX)-derived oxylipins, 11-hydroxy-docosapentaenoic acid and 14-hydroxy-docosapentaenoic acid, to assess their ability to inhibit platelet activation. Pharmacological and genetic approaches were used to elucidate a role for DPA and its oxylipins in preventing platelet activation. DPAn-6 was found to be significantly increased in platelets following fatty acid supplementation, and it potently inhibited platelet activation through its 12-LOX-derived oxylipins. The inhibitory effects were selectively reversed through inhibition of the nuclear receptor peroxisome proliferator activator receptor-α (PPARα). PPARα binding was confirmed using a PPARα transcription reporter assay, as well as PPARα-/- mice. These approaches confirmed that selectivity of platelet inhibition was due to effects of DPA oxylipins acting through PPARα. Mice administered DPAn-6 or its oxylipins exhibited reduced thrombus formation following vessel injury, which was prevented in PPARα-/- mice. Hence, the current study demonstrates that DPAn-6 and its oxylipins potently and effectively inhibit platelet activation and thrombosis following a vascular injury. Platelet function is regulated, in part, through an oxylipin-induced PPARα-dependent manner, suggesting that targeting PPARα may represent an alternative strategy to treat thrombotic-related diseases.
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http://dx.doi.org/10.1182/bloodadvances.2020002493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509882PMC
September 2020

A 12-lipoxygenase-Gpr31 signaling axis is required for pancreatic organogenesis in the zebrafish.

FASEB J 2020 Nov 12;34(11):14850-14862. Epub 2020 Sep 12.

Department of Pediatrics, Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN, USA.

12-Lipoxygenase (12-LOX) is a key enzyme in arachidonic acid metabolism, and alongside its major product, 12-HETE, plays a key role in promoting inflammatory signaling during diabetes pathogenesis. Although 12-LOX is a proposed therapeutic target to protect pancreatic islets in the setting of diabetes, little is known about the consequences of blocking its enzymatic activity during embryonic development. Here, we have leveraged the strengths of the zebrafish-genetic manipulation and pharmacologic inhibition-to interrogate the role of 12-LOX in pancreatic development. Lipidomics analysis during zebrafish development demonstrated that 12-LOX-generated metabolites of arachidonic acid increase sharply during organogenesis stages, and that this increase is blocked by morpholino-directed depletion of 12-LOX. Furthermore, we found that either depletion or inhibition of 12-LOX impairs both exocrine pancreas growth and unexpectedly, the generation of insulin-producing β cells. We demonstrate that morpholino-mediated knockdown of GPR31, a purported G-protein-coupled receptor for 12-HETE, largely phenocopies both the depletion and the inhibition of 12-LOX. Moreover, we show that loss of GPR31 impairs pancreatic bud fusion and pancreatic duct morphogenesis. Together, these data provide new insight into the requirement of 12-LOX in pancreatic organogenesis and islet formation, and additionally provide evidence that its effects are mediated via a signaling axis that includes the 12-HETE receptor GPR31.
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http://dx.doi.org/10.1096/fj.201902308RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606739PMC
November 2020

Fatty Acid Allosteric Regulation of C-H Activation in Plant and Animal Lipoxygenases.

Molecules 2020 Jul 24;25(15). Epub 2020 Jul 24.

Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, USA.

Lipoxygenases (LOXs) catalyze the (per) oxidation of fatty acids that serve as important mediators for cell signaling and inflammation. These reactions are initiated by a C-H activation step that is allosterically regulated in plant and animal enzymes. LOXs from higher eukaryotes are equipped with an N-terminal PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin) domain that has been implicated to bind to small molecule allosteric effectors, which in turn modulate substrate specificity and the rate-limiting steps of catalysis. Herein, the kinetic and structural evidence that describes the allosteric regulation of plant and animal lipoxygenase chemistry by fatty acids and their derivatives are summarized.
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http://dx.doi.org/10.3390/molecules25153374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7436259PMC
July 2020

15-Lipoxygenase-1 biosynthesis of 7S,14S-diHDHA implicates 15-lipoxygenase-2 in biosynthesis of resolvin D5.

J Lipid Res 2020 07 13;61(7):1087-1103. Epub 2020 May 13.

Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064. Electronic address:

The two oxylipins 7S,14S-dihydroxydocosahexaenoic acid (diHDHA) and 7S,17S-diHDHA [resolvin D5 (RvD5)] have been found in macrophages and infectious inflammatory exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of DHA by lipoxygenase (LOX) enzymes, specifically, by human 5-LOX (h5-LOX) first to 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-DHA (7S-HDHA), followed by human platelet 12-LOX (h12-LOX) to form 7(S),14(S)-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-DHA (7S,14S-diHDHA) or human reticulocyte 15-LOX-1 (h15-LOX-1) to form RvD5. In this work, we determined that oxidation of 7(S)-hydroperoxy-4Z,8E,10Z,13Z,16Z,19Z-DHA to 7S,14S-diHDHA is performed with similar kinetics by either h12-LOX or h15-LOX-1. The oxidation at C14 of DHA by h12-LOX was expected, but the noncanonical reaction of h15-LOX-1 to make over 80% 7S,14S-diHDHA was larger than expected. Results of computer modeling suggested that the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of Ile399, forcing the hydrogen abstraction from C12 to oxygenate on C14 but not C17. This result raised questions regarding the synthesis of RvD5. Strikingly, we found that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than does h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages suggests that it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14(S)-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-DHA and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-DHA are kinetically slow compared with DHA, suggesting that these reactions may be minor biosynthetic routes in vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micromolar potencies, which could directly regulate clot resolution.
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http://dx.doi.org/10.1194/jlr.RA120000777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328043PMC
July 2020

Biosynthesis of the Maresin Intermediate, 13S,14S-Epoxy-DHA, by Human 15-Lipoxygenase and 12-Lipoxygenase and Its Regulation through Negative Allosteric Modulators.

Biochemistry 2020 05 7;59(19):1832-1844. Epub 2020 May 7.

Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California 95064, United States.

Human reticulocyte 15-lipoxygenase-1 (h15-LOX-1 or ALOX15) and platelet 12-lipoxygenase (h12-LOX or ALOX12) catalysis of docosahexaenoic acid (DHA) and the maresin precursor, 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-HpDHA), were investigated to determine their product profiles and relative rates in the biosynthesis of the key maresin intermediate, 13S,14S-epoxy-4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid (13S,14S-epoxy-DHA). Both enzymes converted DHA to 14S-HpDHA, with h12-LOX having a 39-fold greater / value (14.0 ± 0.8 s μM) than that of h15-LOX-1 (0.36 ± 0.08 s μM) and a 1.8-fold greater 14S-HpDHA product selectivity, 81 and 46%, respectively. However, h12-LOX was markedly less effective at producing 13S,14S-epoxy-DHA from 14S-HpDHA than h15-LOX-1, with a 4.6-fold smaller / value, 0.0024 ± 0.0002 and 0.11 ± 0.006 s μM, respectively. This is the first evidence of h15-LOX-1 to catalyze this reaction and reveals a novel pathway for maresin biosynthesis. In addition, epoxidation of 14S-HpDHA is negatively regulated through allosteric oxylipin binding to h15-LOX-1 and h12-LOX. For h15-LOX-1, 14S-HpDHA ( = 6.0 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) ( = 3.5 μM), and 14S-hydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic acid (14S-HDPA) ( = 4.0 μM) were shown to decrease 13S,14S-epoxy-DHA production. h12-LOX was also shown to be allosterically regulated by 14S-HpDHA ( = 3.5 μM) and 14S-HDPA ( = 4.0 μM); however, 12S-HETE showed no effect, indicating for the first time an allosteric response by h12-LOX. Finally, 14S-HpDHA inhibited platelet aggregation at a submicrololar concentration, which may have implications in the benefits of diets rich in DHA. These biosynthetic pathways may help guide maresin biosynthetic investigations and possibly direct therapeutic interventions.
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http://dx.doi.org/10.1021/acs.biochem.0c00233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729281PMC
May 2020

Contributions of 12/15-Lipoxygenase to Bleeding in the Brain Following Ischemic Stroke.

Adv Exp Med Biol 2019 ;1161:125-131

Neuroprotection Research Laboratories, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.

Ischemic strokes are caused by one or more blood clots that typically obstruct one of the major arteries in the brain, but frequently also result in leakage of the blood-brain barrier and subsequent hemorrhage. While it has long been known that the enzyme 12/15-lipoxygenase (12/15-LOX) is up-regulated following ischemic strokes and contributes to neuronal cell death, recent research has shown an additional major role for 12/15-LOX in causing this hemorrhagic transformation. These findings have important implications for the use of 12/15-LOX inhibitors in the treatment of stroke.
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http://dx.doi.org/10.1007/978-3-030-21735-8_12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278041PMC
October 2019

5 S,15 S-Dihydroperoxyeicosatetraenoic Acid (5,15-diHpETE) as a Lipoxin Intermediate: Reactivity and Kinetics with Human Leukocyte 5-Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-Lipoxygenase-1.

Biochemistry 2018 12 15;57(48):6726-6734. Epub 2018 Nov 15.

Department of Chemistry and Biochemistry , University of California, Santa Cruz , 1156 High Street , Santa Cruz , California 95064 , United States.

The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB. For 12-LOX and 5,15-diHpETE, the kinetic parameters are k = 0.17 s and k/ K = 0.011 μM s [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are k = 4.6 s and k/ K = 0.21 μM s (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC of 1.3 μM; however, LXB did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA and LXB are produced by 12-LOX but only if LTA is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].
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http://dx.doi.org/10.1021/acs.biochem.8b00889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270142PMC
December 2018

N-acetylcysteine targets 5 lipoxygenase-derived, toxic lipids and can synergize with prostaglandin E to inhibit ferroptosis and improve outcomes following hemorrhagic stroke in mice.

Ann Neurol 2018 12 29;84(6):854-872. Epub 2018 Nov 29.

Sperling Center for Hemorrhagic Stroke Recovery, Burke Neurological Institute, White Plains, NY.

Objectives: N-acetylcysteine (NAC) is a clinically approved thiol-containing redox modulatory compound currently in trials for many neurological and psychiatric disorders. Although generically labeled as an "antioxidant," poor understanding of its site(s) of action is a barrier to its use in neurological practice. Here, we examined the efficacy and mechanism of action of NAC in rodent models of hemorrhagic stroke.

Methods: Hemin was used to model ferroptosis and hemorrhagic stroke in cultured neurons. Striatal infusion of collagenase was used to model intracerebral hemorrhage (ICH) in mice and rats. Chemical biology, targeted lipidomics, arachidonate 5-lipoxygenase (ALOX5) knockout mice, and viral-gene transfer were used to gain insight into the pharmacological targets and mechanism of action of NAC.

Results: NAC prevented hemin-induced ferroptosis by neutralizing toxic lipids generated by arachidonate-dependent ALOX5 activity. NAC efficacy required increases in glutathione and is correlated with suppression of reactive lipids by glutathione-dependent enzymes such as glutathione S-transferase. Accordingly, its protective effects were mimicked by chemical or molecular lipid peroxidation inhibitors. NAC delivered postinjury reduced neuronal death and improved functional recovery at least 7 days following ICH in mice and can synergize with clinically approved prostaglandin E (PGE ).

Interpretation: NAC is a promising, protective therapy for ICH, which acted to inhibit toxic arachidonic acid products of nuclear ALOX5 that synergized with exogenously delivered protective PGE in vitro and in vivo. The findings provide novel insight into a target for NAC, beyond the generic characterization as an antioxidant, resulting in neuroprotection and offer a feasible combinatorial strategy to optimize efficacy and safety in dosing of NAC for treatment of neurological disorders involving ferroptosis such as ICH. Ann Neurol 2018;84:854-872.
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http://dx.doi.org/10.1002/ana.25356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519209PMC
December 2018

Pseudomonas aeruginosa utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium.

J Clin Invest 2018 10 10;128(10):4639-4653. Epub 2018 Sep 10.

Department of Environmental and Occupational Health and Center for Free Radical and Antioxidant Health and.

Ferroptosis is a death program executed via selective oxidation of arachidonic acid-phosphatidylethanolamines (AA-PE) by 15-lipoxygenases. In mammalian cells and tissues, ferroptosis has been pathogenically associated with brain, kidney, and liver injury/diseases. We discovered that a prokaryotic bacterium, Pseudomonas aeruginosa, that does not contain AA-PE can express lipoxygenase (pLoxA), oxidize host AA-PE to 15-hydroperoxy-AA-PE (15-HOO-AA-PE), and trigger ferroptosis in human bronchial epithelial cells. Induction of ferroptosis by clinical P. aeruginosa isolates from patients with persistent lower respiratory tract infections was dependent on the level and enzymatic activity of pLoxA. Redox phospholipidomics revealed elevated levels of oxidized AA-PE in airway tissues from patients with cystic fibrosis (CF) but not with emphysema or CF without P. aeruginosa. We believe that the evolutionarily conserved mechanism of pLoxA-driven ferroptosis may represent a potential therapeutic target against P. aeruginosa-associated diseases such as CF and persistent lower respiratory tract infections.
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http://dx.doi.org/10.1172/JCI99490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159971PMC
October 2018

12-HETrE inhibits platelet reactivity and thrombosis in part through the prostacyclin receptor.

Blood Adv 2017 Jun 23;1(15):1124-1131. Epub 2017 Jun 23.

Department of Pharmacology, University of Michigan, Ann Arbor, MI.

The dihomo-γ-linolenic acid (DGLA)-derived metabolite of 12-lipoxygenase, 12-hydroxy-eicosatrienoic acid (12-HETrE), was recently shown to potently inhibit thrombus formation without prolonging bleeding in murine models. Although 12-HETrE was found to inhibit platelet activation via the Gα signaling pathway, the Gα-coupled receptor by which 12-HETrE mediates its antiplatelet effects has yet to be identified. Defining the receptor by which 12-HETrE exerts its effects is key to determining its therapeutic potential as an antiplatelet drug. Therefore, the goal of this study was to determine the Gα-coupled platelet receptor through which 12-HETrE exerts its antiplatelet effects. In this study, we showed that pharmacological inhibition of the prostacyclin (IP) receptor in human platelets or genetic ablation of IP in murine platelets prevented 12-HETrE from blocking aggregation in vitro. Furthermore, the antithrombotic effects of 12-HETrE were significantly diminished in IP knockout mice in vivo. Together these data demonstrate that the antiplatelet effects of 12-HETrE are at least partially dependent on IP signaling. Importantly, this work identified 12-HETrE as a novel regulator of IP signaling that may aid in the rationale for design of novel therapeutics to inhibit platelet function. Additionally, this study provides further insight into the mechanism by which DGLA supplementation inhibits platelets function.
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http://dx.doi.org/10.1182/bloodadvances.2017006155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728320PMC
June 2017

PEBP1 Wardens Ferroptosis by Enabling Lipoxygenase Generation of Lipid Death Signals.

Cell 2017 Oct;171(3):628-641.e26

Department of Environmental and Occupational Health, Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA, USA; Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA; Department of Radiation Oncology, University of Pittsburgh, Pittsburgh, PA, USA. Electronic address:

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.
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http://dx.doi.org/10.1016/j.cell.2017.09.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5683852PMC
October 2017

First Selective 12-LOX Inhibitor, ML355, Impairs Thrombus Formation and Vessel Occlusion In Vivo With Minimal Effects on Hemostasis.

Arterioscler Thromb Vasc Biol 2017 10 3;37(10):1828-1839. Epub 2017 Aug 3.

From the Department of Pharmacology (R.A., B.E.T., K.M., J.Y., M.H.) and Department of Internal Medicine, Division of Cardiovascular Medicine (M.H.), University of Michigan, Ann Arbor; Chemistry and Biochemistry, University of California Santa Cruz (J.C.F., A.G., T.R.H.); and National Institutes of Health Chemical Genomics Center, National Center for Advancing Translational Sciences, Rockville, MD (D.K.L., A.J., A.S., D.J.M.).

Objective: Adequate platelet reactivity is required for maintaining hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi. Platelet 12(S)-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated to regulate platelet function and thrombosis ex vivo, supporting a key role for 12-LOX in the regulation of in vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Here, we studied the effect of the first highly selective 12-LOX inhibitor, ML355, on in vivo thrombosis and hemostasis.

Approach And Results: ML355 dose-dependently inhibited human platelet aggregation and 12-LOX oxylipin production, as confirmed by mass spectrometry. Interestingly, the antiplatelet effects of ML355 were reversed after exposure to high concentrations of thrombin in vitro. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen were attenuated in whole blood treated with ML355 comparable to aspirin. Oral administration of ML355 in mice showed reasonable plasma drug levels by pharmacokinetic assessment. ML355 treatment impaired thrombus growth and vessel occlusion in FeCl-induced mesenteric and laser-induced cremaster arteriole thrombosis models in mice. Importantly, hemostatic plug formation and bleeding after treatment with ML355 was minimal in mice in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model.

Conclusions: Our data strongly support 12-LOX as a key determinant of platelet reactivity in vivo, and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapy.
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http://dx.doi.org/10.1161/ATVBAHA.117.309868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620123PMC
October 2017

12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gαs Signaling in Platelets.

Arterioscler Thromb Vasc Biol 2016 10 28;36(10):2068-77. Epub 2016 Jul 28.

From the Department of Pharmacology (J.Y., B.E.T., R.A., M.H.) and Department of Internal Medicine, Division of Cardiovascular Medicine (M.H.), University of Michigan, Ann Arbor; Cardeza Foundation for Hematological Research, Thomas Jefferson University, Philadelphia, PA (J.Y., B.E.T., R.A., P.F.-P., J.Y., M.H.); and Department of Chemistry and Biochemistry, University of California Santa Cruz (A.R.G., C.J.F., T.R.H.).

Objective: Dietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis.

Approach And Results: DGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX(-/-)). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX(-/-) mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked G-protein-coupled receptor pathway in human platelets.

Conclusions: This study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit-linked G-protein-coupled receptor-dependent manner.
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http://dx.doi.org/10.1161/ATVBAHA.116.308050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5488693PMC
October 2016

Azole Antifungal Sensitivity of Sterol 14α-Demethylase (CYP51) and CYP5218 from Malassezia globosa.

Sci Rep 2016 06 13;6:27690. Epub 2016 Jun 13.

Centre for Cytochrome P450 Biodiversity, Institute of Life Science, Swansea University Medical School, Swansea, Wales SA2 8PP, United Kingdom.

Malassezia globosa cytochromes P450 CYP51 and CYP5218 are sterol 14α-demethylase (the target of azole antifungals) and a putative fatty acid metabolism protein (and a potential azole drug target), respectively. Lanosterol, eburicol and obtusifoliol bound to CYP51 with Kd values of 32, 23 and 28 μM, respectively, catalyzing sterol 14α-demethylation with respective turnover numbers of 1.7 min(-1), 5.6 min(-1) and 3.4 min(-1). CYP5218 bound a range of fatty acids with linoleic acid binding strongest (Kd 36 μM), although no metabolism could be detected in reconstitution assays or role in growth on lipids. Clotrimazole, fluconazole, itraconazole, ketoconazole, voriconazole and ketaminazole bound tightly to CYP51 (Kd ≤ 2 to 11 nM). In contrast, fluconazole did not bind to CYP5218, voriconazole and ketaminazole bound weakly (Kd ~107 and ~12 μM), whereas ketoconazole, clotrimazole and itraconazole bound strongest to CYP5218 (Kd ~1.6, 0.5 and 0.4 μM) indicating CYP5218 to be only a secondary target of azole antifungals. IC50 determinations confirmed M. globosa CYP51 was strongly inhibited by azole antifungals (0.15 to 0.35 μM). MIC100 studies showed itraconazole should be considered as an alternative to ketoconazole given the potency and safety profiles and the CYP51 assay system can be used in structure-activity studies in drug development.
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http://dx.doi.org/10.1038/srep27690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4904373PMC
June 2016

Biochemical and Cellular Characterization and Inhibitor Discovery of Pseudomonas aeruginosa 15-Lipoxygenase.

Biochemistry 2016 06 3;55(23):3329-40. Epub 2016 Jun 3.

Department of Chemistry and Biochemistry, University of California , Santa Cruz, California 95064, United States.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial and chronic infections in immunocompromised patients. P. aeruginosa secretes a lipoxygenase, LoxA, but the biological role of this enzyme is currently unknown. LoxA is poorly similar in sequence to both soybean LOX-1 (s15-LOX-1) and human 15-LOX-1 (37 and 39%, respectively) yet has kinetics comparably fast versus those of s15-LOX-1 (at pH 6.5, Kcat = 181 ± 6 s(-1) and Kcat/KM = 16 ± 2 μM(-1) s(-1)). LoxA is capable of efficiently catalyzing the peroxidation of a broad range of free fatty acid (FA) substrates (e.g., AA and LA) with high positional specificity, indicating a 15-LOX. Its mechanism includes hydrogen atom abstraction [a kinetic isotope effect (KIE) of >30], yet LoxA is a poor catalyst against phosphoester FAs, suggesting that LoxA is not involved in membrane decomposition. LoxA also does not react with 5- or 15-HETEs, indicating poor involvement in lipoxin production. A LOX high-throughput screen of the LOPAC library yielded a variety of low-micromolar inhibitors; however, none selectively targeted LoxA over the human LOX isozymes. With respect to cellular activity, the level of LoxA expression is increased when P. aeruginosa undergoes the transition to a biofilm mode of growth, but LoxA is not required for biofilm growth on abiotic surfaces. However, LoxA does appear to be required for biofilm growth in association with the host airway epithelium, suggesting a role for LoxA in mediating bacterium-host interactions during colonization.
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http://dx.doi.org/10.1021/acs.biochem.6b00338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249612PMC
June 2016

Strict Regiospecificity of Human Epithelial 15-Lipoxygenase-2 Delineates Its Transcellular Synthesis Potential.

Biochemistry 2016 05 13;55(20):2832-40. Epub 2016 May 13.

Department Chemistry and Biochemistry, University of California at Santa Cruz , 1156 High Street, Santa Cruz, California 95064, United States.

Lipoxins are an important class of lipid mediators that induce the resolution of inflammation and arise from transcellular exchange of arachidonic acid (AA)-derived lipoxygenase products. Human epithelial 15-lipoxygenase-2 (h15-LOX-2), the major lipoxygenase in macrophages, has exhibited strict regiospecificity, catalyzing only the hydroperoxidation of carbon 15 of AA. To determine the catalytic potential of h15-LOX-2 in transcellular synthesis events, we reacted it with the three lipoxygenase-derived monohydroperoxy-eicosatetraenoic acids (HPETE) in humans: 5-HPETE, 12-HPETE, and 15-HPETE. Only 5-HPETE was a substrate for h15-LOX-2, and the steady-state catalytic efficiency (kcat/Km) of this reaction was 31% of the kcat/Km of AA. The only major product of h15-LOX-2's reaction with 5-HPETE was the proposed lipoxin intermediate, 5,15-dihydroperoxy-eicosatetraenoic acid (5,15-diHPETE). However, h15-LOX-2 did not react further with 5,15-diHPETE to produce lipoxins. This result is consistent with the specificity of h15-LOX-2 despite the increased reactivity of 5,15-diHPETE. Density functional theory calculations determined that the radical, after abstracting the C10 hydrogen atom from 5,15-diHPETE, had an energy 5.4 kJ/mol lower than that of the same radical generated from AA, demonstrating the facility of 5,15-diHPETE to form lipoxins. Interestingly, h15-LOX-2 does react with 5S,6R-diHETE, forming LipoxinA4, indicating the gemdiol does not prohibit h15-LOX-2 reactivity. Taken together, these results demonstrate the strict regiospecificity of h15-LOX-2 that circumscribes its role in transcellular synthesis.
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http://dx.doi.org/10.1021/acs.biochem.5b01339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657383PMC
May 2016

Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models.

Sci Transl Med 2016 Mar;8(328):328ra29

Sperling Center for Hemorrhagic Stroke Recovery, Burke Medical Research Institute, White Plains, NY 10605, USA. Feil Family Brain and Mind Research Institute, Weill Medical College of Cornell University, New York, NY 10065, USA.

Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier-permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models.
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http://dx.doi.org/10.1126/scitranslmed.aac6008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341138PMC
March 2016

A potent and selective inhibitor targeting human and murine 12/15-LOX.

Bioorg Med Chem 2016 Mar 21;24(6):1183-90. Epub 2016 Jan 21.

Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95060, United States. Electronic address:

Human reticulocyte 12/15-lipoxygenase (h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h12/15-LOX is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood-brain barrier leakage. The discovery of inhibitors that selectively target recombinant h12/15-LOX in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. Herein, we report a new family of inhibitors discovered in a High Throughput Screen (HTS) that are selective and potent against recombinant h12/15-LOX and cellular mouse 12/15-LOX (m12/15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC50 potency of 3.4±0.5 μM against h12/15-LOX in vitro and an ex vivo IC50 potency of approximately 10 μM in a mouse neuronal cell line, HT-22. Compound 99089 displays greater than 30-fold selectivity versus h5-LOX and COX-2, 15-fold versus h15-LOX-2 and 10-fold versus h12-LOX, when tested at 20 μM inhibitor concentration. Steady-state inhibition kinetics reveals that the mode of inhibition of 99089 against h12/15-LOX is that of a mixed inhibitor with a Kic of 1.0±0.08 μM and a Kiu of 6.0±3.3 μM. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h12/15-LOX in vitro and m12/15-LOX ex vivo.
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http://dx.doi.org/10.1016/j.bmc.2016.01.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4778748PMC
March 2016

Minireview: 12-Lipoxygenase and Islet β-Cell Dysfunction in Diabetes.

Mol Endocrinol 2015 Jun 24;29(6):791-800. Epub 2015 Mar 24.

Departments of Pediatrics and the Center for Diabetes and Metabolic Diseases (S.A.T., R.G.M.), Biochemistry and Molecular Biology (E.B., R.G.M.), Medicine (R.G.M.), and Cellular and Integrative Physiology (R.G.M.), Indiana University School of Medicine, Indianapolis, Indiana 46202; Department of Chemistry and Biochemistry (T.R.H.), University of California, Santa Cruz, Santa Cruz, California 95064; National Center for Advancing Translational Sciences (D.J.M.), National Institutes of Health, Rockville, Maryland 20850; and Department of Medicine and the Strelitz Diabetes Center (J.L.N.), Eastern Virginia Medical School, Norfolk, Virginia 23507.

The insulin producing islet β-cells have increasingly gained attention for their role in the pathogeneses of virtually all forms of diabetes. Dysfunction, de-differentiation, and/or death of β-cells are pivotal features in the transition from normoglycemia to hyperglycemia in both animal models of metabolic disease and humans. In both type 1 and type 2 diabetes, inflammation appears to be a central cause of β-cell derangements, and molecular pathways that modulate inflammation or the inflammatory response are felt to be prime targets of future diabetes therapy. The lipoxygenases (LOs) represent a class of enzymes that oxygenate cellular polyunsaturated fatty acids to produce inflammatory lipid intermediates that directly and indirectly affect cellular function and survival. The enzyme 12-LO is expressed in all metabolically active tissues, including pancreatic islets, and has received increasing attention for its role in promoting cellular inflammation in the setting of diabetes. Genetic deletion models of 12-LO in mice reveal striking protection from metabolic disease and its complications and an emerging body of literature has implicated its role in human disease. This review focuses on the evidence supporting the proinflammatory role of 12-LO as it relates to islet β-cells, and the potential for 12-LO inhibition as a future avenue for the prevention and treatment of metabolic disease.
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http://dx.doi.org/10.1210/me.2015-1041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447641PMC
June 2015

A high-throughput mass spectrometric assay for discovery of human lipoxygenase inhibitors and allosteric effectors.

Anal Biochem 2015 May 21;476:45-50. Epub 2015 Feb 21.

Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA 95064, USA. Electronic address:

Lipoxygenases (LOXs) regulate inflammation through the production of a variety of molecules whose specific downstream effects are not entirely understood due to the complexity of the inflammation pathway. The generation of these biomolecules can potentially be inhibited and/or allosterically regulated by small synthetic molecules. The current work describes the first mass spectrometric high-throughput method for identifying small molecule LOX inhibitors and LOX allosteric effectors that change the substrate preference of human lipoxygenase enzymes. Using a volatile buffer and an acid-labile detergent, enzymatic products can be directly detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS) without the need for organic extraction. The method also reduces the required enzyme concentration compared with traditional ultraviolet (UV) absorbance methods by approximately 30-fold, allowing accurate binding affinity measurements for inhibitors with nanomolar affinity. The procedure was validated using known LOX inhibitors and the allosteric effector 13(S)-hydroxy-9Z,11E-octadecadienoic acid (13-HODE).
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http://dx.doi.org/10.1016/j.ab.2015.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4398643PMC
May 2015

The potential of 12/15-lipoxygenase inhibitors in stroke therapy.

Future Med Chem 2014 ;6(17):1853-5

Neuroprotection Research Laboratory, Dept. of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.

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http://dx.doi.org/10.4155/fmc.14.129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280907PMC
August 2015

Selective inhibition of 12-lipoxygenase protects islets and beta cells from inflammatory cytokine-mediated beta cell dysfunction.

Diabetologia 2015 Mar 23;58(3):549-57. Epub 2014 Nov 23.

Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, 700 W. Olney Road, Norfolk, VA, 23507, USA,

Aims/hypothesis: Islet inflammation leads to loss of functional pancreatic beta cell mass. Increasing evidence suggests that activation of 12-lipoxygenase leads to inflammatory beta cell loss. This study evaluates new specific small-molecule inhibitors of 12-lipoxygenase for protecting rodent and human beta cells from inflammatory damage.

Methods: Mouse beta cell lines and mouse and human islets were treated with inflammatory cytokines IL-1β, TNFα and IFNγ in the absence or presence of novel selective 12-lipoxygenase inhibitors. Glucose-stimulated insulin secretion (GSIS), gene expression, cell survival and 12-S-hydroxyeicosatetraenoic acid (12-S-HETE) levels were evaluated using established methods. Pharmacokinetic analysis was performed with the lead inhibitor in CD1 mice.

Results: Inflammatory cytokines led to the loss of human beta cell function, elevated cell death, increased inflammatory gene expression and upregulation of 12-lipoxygenase expression and activity (measured by 12-S-HETE generation). Two 12-lipoxygenase inhibitors, Compounds 5 and 9, produced a concentration-dependent reduction of stimulated 12-S-HETE levels. GSIS was preserved in the presence of the 12-lipoxygenase inhibitors. 12-Lipoxygenase inhibition preserved survival of primary mouse and human islets. When administered orally, Compound 5 reduced plasma 12-S-HETE in CD1 mice. Compounds 5 and 9 preserved the function and survival of human donor islets exposed to inflammatory cytokines.

Conclusions/interpretation: Selective inhibition of 12-lipoxygenase activity confers protection to beta cells during exposure to inflammatory cytokines. These concept validation studies identify 12-lipoxygenase as a promising target in the prevention of loss of functional beta cells in diabetes.
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http://dx.doi.org/10.1007/s00125-014-3452-0DOI Listing
March 2015

Enzymatic Studies of Isoflavonoids as Selective and Potent Inhibitors of Human Leukocyte 5-Lipo-Oxygenase.

Chem Biol Drug Des 2015 Jul 28;86(1):114-21. Epub 2014 Nov 28.

Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, 95064.

Continuing our search to find more potent and selective 5-LOX inhibitors, we present now the enzymatic evaluation of seventeen isoflavones (IR) and nine isoflavans (HIR), and their in vitro and in cellulo potency against human leukocyte 5-LOX. Of the 26 compounds tested, 10 isoflavones and 9 isoflavans possessed micromolar potency, but only three were selective against 5-LOX (IR-2, HIR-303, and HIR-309), with IC50 values at least 10 times lower than those of 12-LOX, 15-LOX-1, and 15-LOX-2. Of these three, IR-2 (6,7-dihydroxy-4-methoxy-isoflavone, known as texasin) was the most selective 5-LOX inhibitor, with over 80-fold potency difference compared with other isozymes; Steered Molecular Dynamics (SMD) studies supported these findings. The presence of the catechol group on ring A (6,7-dihydroxy versus 7,8-dihydroxy) correlated with their biological activity, but the reduction of ring C, converting the isoflavones to isoflavans, and the substituent positions on ring B did not affect their potency against 5-LOX. Two of the most potent/selective inhibitors (HIR-303 and HIR-309) were reductive inhibitors and were potent against 5-LOX in human whole blood, indicating that isoflavans can be potent and selective inhibitors against human leukocyte 5-LOX in vitro and in cellulo.
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http://dx.doi.org/10.1111/cbdd.12469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270146PMC
July 2015

A high throughput screen identifies potent and selective inhibitors to human epithelial 15-lipoxygenase-2.

PLoS One 2014 11;9(8):e104094. Epub 2014 Aug 11.

Chemistry and Biochemistry Department, University of California Santa Cruz, Santa Cruz, California, United States of America.

Lipoxygenase (LOX) enzymes catalyze the hydroperoxidation of arachidonic acid and other polyunsaturated fatty acids to hydroxyeicosatetraenoic acids with varying positional specificity to yield important biological signaling molecules. Human epithelial 15-lipoxygenase-2 (15-LOX-2) is a highly specific LOX isozyme that is expressed in epithelial tissue and whose activity has been correlated with suppression of tumor growth in prostate and other epithelial derived cancers. Despite the potential utility of an inhibitor to probe the specific role of 15-LOX-2 in tumor progression, no such potent/specific 15-LOX-2 inhibitors have been reported to date. This study employs high throughput screening to identify two novel, specific 15-LOX-2 inhibitors. MLS000545091 is a mixed-type inhibitor of 15-LOX-2 with a Ki of 0.9+/-0.4 µM and has a 20-fold selectivity over 5-LOX, 12-LOX, 15-LOX-1, COX-1, and COX-2. MLS000536924 is a competitive inhibitor with a Ki of 2.5+/-0.5 µM and also possesses 20-fold selectivity toward 15-LOX-2 over the other oxygenases, listed above. Finally, neither compound possesses reductive activity towards the active-site ferrous ion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104094PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128814PMC
April 2015

Platelet 12-LOX is essential for FcγRIIa-mediated platelet activation.

Blood 2014 Oct 6;124(14):2271-9. Epub 2014 Aug 6.

Cardeza Foundation for Hematologic Research, Department of Medicine, Thomas Jefferson University, Philadelphia, PA;

Platelets are essential in maintaining hemostasis following inflammation or injury to the vasculature. Dysregulated platelet activity often results in thrombotic complications leading to myocardial infarction and stroke. Activation of the FcγRIIa receptor leads to immune-mediated thrombosis, which is often life threatening in patients undergoing heparin-induced thrombocytopenia or sepsis. Inhibiting FcγRIIa-mediated activation in platelets has been shown to limit thrombosis and is the principal target for prevention of immune-mediated platelet activation. In this study, we show for the first time that platelet 12(S)-lipoxygenase (12-LOX), a highly expressed oxylipin-producing enzyme in the human platelet, is an essential component of FcγRIIa-mediated thrombosis. Pharmacologic inhibition of 12-LOX in human platelets resulted in significant attenuation of FcγRIIa-mediated aggregation. Platelet 12-LOX was shown to be essential for FcγRIIa-induced phospholipase Cγ2 activity leading to activation of calcium mobilization, Rap1 and protein kinase C activation, and subsequent activation of the integrin αIIbβ3. Additionally, platelets from transgenic mice expressing human FcγRIIa but deficient in platelet 12-LOX, failed to form normal platelet aggregates and exhibited deficiencies in Rap1 and αIIbβ3 activation. These results support an essential role for 12-LOX in regulating FcγRIIa-mediated platelet function and identifies 12-LOX as a potential therapeutic target to limit immune-mediated thrombosis.
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http://dx.doi.org/10.1182/blood-2014-05-575878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183986PMC
October 2014

Inhibitory and mechanistic investigations of oxo-lipids with human lipoxygenase isozymes.

Bioorg Med Chem 2014 Aug 21;22(15):4293-7. Epub 2014 May 21.

Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95064, United States. Electronic address:

Oxo-lipids, a large family of oxidized human lipoxygenase (hLOX) products, are of increasing interest to researchers due to their involvement in different inflammatory responses in the cell. Oxo-lipids are unique because they contain electrophilic sites that can potentially form covalent bonds through a Michael addition mechanism with nucleophilic residues in protein active sites and thus increase inhibitor potency. Due to the resemblance of oxo-lipids to LOX substrates, the inhibitor potency of 4 different oxo-lipids; 5-oxo-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5-oxo-ETE), 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE), 12-oxo-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid (12-oxo-ETE), and 13-oxo-9,11-(Z,E)-octadecadienoic acid (13-oxo-ODE) were determined against a library of LOX isozymes; leukocyte 5-lipoxygenase (h5-LOX), human reticulocyte 15-lipoxygenase-1 (h15-LOX-1), human platelet 12-lipoxygenase (h12-LOX), human epithelial 15-lipoxygenase-2 (h15-LOX-2), soybean 15-lipoxygenase-1 (s15-LOX-1), and rabbit reticulocyte 15-LOX (r15-LOX). 15-Oxo-ETE exhibited the highest potency against h12-LOX, with an IC₅₀=1 ± 0.1 μM and was highly selective. Steady state inhibition kinetic experiments determined 15-oxo-ETE to be a mixed inhibitor against h12-LOX, with a Kic value of 0.087 ± 0.008 μM and a Kiu value of 2.10 ± 0.8 μM. Time-dependent studies demonstrated irreversible inhibition with 12-oxo-ETE and h15-LOX-1, however, the concentration of 12-oxo-ETE required (Ki=36.8 ± 13.2 μM) and the time frame (k₂=0.0019 ± 0.00032 s(-1)) were not biologically relevant. These data are the first observations that oxo-lipids can inhibit LOX isozymes and may be another mechanism in which LOX products regulate LOX activity.
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http://dx.doi.org/10.1016/j.bmc.2014.05.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112157PMC
August 2014