Human 5-lipoxygenase Research Articles

1469-P: Inhibition of the Integrated Stress Response Increases PD-L1 in Islet ß-Cells and Protects against Autoimmune Diabetes

Type 1 diabetes (T1D) has become viewed as an autoimmune disease initiated and propagated by stress responses in islet β cells. The integrated stress response (ISR) is activated upon inflammatory signaling and delimits protein production through the actions of phospho-eIF2α. We hypothesized that inhibition of the β cell ISR will protect against β cell immunogenicity and development of T1D. To test this hypothesis, we studied the effect of ISR inhibition using ISRIB (p-eIF2α inhibitor) and VLX-10 (human 12-lipoxygenase inhibitor) on the response of isolated human islets and EndoC-βH1 cells to inflammatory cytokines (IFN-γ+IL-1β) and studied the effect of ISR inhibition on T1D outcomes in NOD mice. Human islets exposed to cytokines exhibited an increase in p-eIF2α and suppression of mRNA translation initiation (by polysome profiling) , consistent with activation of the ISR. Inhibition of the ISR with ISRIB or VLX-10promoted production of PD-L1, an immune checkpoint protein that suppresses the immune response, in human islets and EndoC-βH1 cells. This finding suggested that blockade of the ISR in β cells might thwart immune attack. To assess if inhibition of the ISR using the human-specific 12-lipoxygenase inhibitor VLX-10modifies T1D progression, we generated mice in which the gene encoding mouse 12-lipoxygenase gene was replaced by the corresponding human gene. These “humanized” mice were subsequently backcrossed onto the NOD background, then treated in the prediabetic phase with VLX-1005. Male and female humanized mice developed T1D at the expected frequency, but those receiving VLX-10showed significant delay of T1D onset. Consistent with the suppression of autoimmunity, insulitis was significantly reduced and β cell mass was significantly increased in treated animals. Our results support the targeting of the ISR through inhibition of the 12-lipoxygenase enzyme provides an opportunity for the modification of T1D disease progression. Disclosure F.Huang: None. A.Kulkarni: None. F.Syed: None. D.J.Maloney: Employee; Veralox Therapeutics. S.A.Tersey: n/a. R.G.Mirmira: None. Funding NIH (UDK127786-02)

Non‐Competitive Inhibition by a Substrate Mimetic of Human 5‐Lipoxygenase

Human 5‐lipoxygenase (5‐LOX) initiates the synthesis of the leukotrienes (LT) through a two‐step reaction: addition of molecular oxygen to arachidonic acid (AA) into 5–hydroperoxyeicosatetraenoic acid (5‐HPETE) and hydrolysis of 5‐HPETE into leukotriene A4. LTs are highly potent, pro‐inflammatory mediators, which have been implicated in asthmatic and allergic responses. 5‐LOX is mobilized in leukocytes by Ca2+ binding and targets to the membrane to receive substrate from its helper protein, 5‐lipoxygenase‐activating protein (FLAP). These allosteric partners are unnecessary in in vitro experiments but enhance activity in intact cells. The crystal structure of 5‐LOX was revealed in a “closed conformation” with the catalytic iron inaccessible to surface solvent. This closed active site is the result of a unique conformation of helix alpha 2 (Hα2) where two aromatic residues plug the putative substrate‐access portal. 5‐LOX has a disjointed Hα2 of 3 turns connected with multiple loop‐to‐helix transitions permitting this plugged conformation of the active site. For comparison, the structure of another human isoform, 15‐LOX‐2, has Hα2 in a fully elongated conformation of 6 turns and the catalytic iron solvent accessible for substrate entry. The detergent C8E4 was co‐crystallized with 15‐LOX‐2 in the active site, kinetically identified as a competitive inhibitor, and is an excellent substrate mimetic of AA. Initial kinetic studies of 5‐LOX with C8E4 have shown non‐competitive inhibition patterns. We detailed a non‐Michaelis‐Menton kinetic response of 5‐LOX to increasing amounts of substrate at the last EB meeting (https://doi.org/10.1096/fasebj.2020.34.s1.01876). We hypothesized that a ligand‐induced slow transition of the conformation of Hα2 causes the sigmoidal response to substrate by the monomeric enzyme. Kinetic cooperativity of monomeric enzymes with single substrate‐binding sites, while rare, is represented in highly regulated enzymes. We wanted to further elaborate on this model by performing inhibition kinetics of a substrate mimetic of AA to 5‐LOX. Non‐competitive inhibition of an active site binder is further evidence of a slow conformational change, as it indicates a two‐step inhibition mechanism with the slow transition to an inactive enzyme state. Understanding the inhibition kinetics of an enzyme that adopts multiple conformations and detailing the molecular determinants responsible for inhibitor binding could help delineate some of the discrepancies observed for different 5‐LOX inhibitor potencies in different cellular environments. Anti‐leukotriene therapies has proven to be a successful avenue for lowering inflammation in many disease states and more informed structure‐based drug design approach could hasten their development.

In Vitro Inhibition of Lactobacillus Plantarum 1 from Fermented Coconut Toddy against Human 5-Lipoxygenase

In Vitro Inhibition of Lactobacillus Plantarum 1 from Fermented Coconut Toddy against Human 5-Lipoxygenase

Inhibitors of Human 5-Lipoxygenase Potently Interfere With Prostaglandin Transport.

5-Lipoxygenase (5-LO) is the key enzyme in the formation of pro-inflammatory leukotrienes (LT) which play an important role in a number of inflammatory diseases. Accordingly, 5-LO inhibitors are frequently used to study the role of 5-LO and LT in models of inflammation and cancer. Interestingly, the therapeutic efficacy of these inhibitors is highly variable. Here we show that the frequently used 5-LO inhibitors AA-861, BWA4C, C06, CJ-13,610 and the FDA approved compound zileuton as well as the pan-LO inhibitor nordihydroguaiaretic acid interfere with prostaglandin E2 (PGE2) release into the supernatants of cytokine-stimulated (TNFα/IL-1β) HeLa cervix carcinoma, A549 lung cancer as well as HCA-7 colon carcinoma cells with similar potencies compared to their LT inhibitory activities (IC50 values ranging from 0.1–9.1 µM). In addition, AA-861, BWA4C, CJ-13,610 and zileuton concentration-dependently inhibited bacterial lipopolysaccharide triggered prostaglandin (PG) release into human whole blood. Western Blot analysis revealed that inhibition of expression of enzymes involved in PG synthesis was not part of the underlying mechanism. Also, liberation of arachidonic acid which is the substrate for PG synthesis as well as PGH2 and PGE2 formation were not impaired by the compounds. However, accumulation of intracellular PGE2 was found in the inhibitor treated HeLa cells suggesting inhibition of PG export as major mechanism. Further, experiments showed that the PG exporter ATP-binding cassette transporter multidrug resistance protein 4 (MRP-4) is targeted by the inhibitors and may be involved in the 5-LO inhibitor-mediated PGE2 inhibition. In conclusion, the pharmacological effects of a number of 5-LO inhibitors are compound-specific and involve the potent inhibition of PGE2 export. Results from experimental models on the role of 5-LO in inflammation and pain using 5-LO inhibitors may be misleading and their use as pharmacological tools in experimental models has to be revisited. In addition, 5-LO inhibitors may serve as new scaffolds for the development of potent prostaglandin export inhibitors.

On the biosynthesis of specialized pro-resolving mediators in human neutrophils and the influence of cell integrity

Neutrophils are key players in inflammation initiation and resolution. Little attention has been paid to the detailed biosynthesis of specialized pro-resolving mediators (SPM) in these cells. We investigated SPM formation in human polymorphonuclear leukocytes (PMNL), in broken PMNL preparations and recombinant human 5-lipoxygenase (5-LO) supplemented with the SPM precursor lipids 15-Hydroxyeicosatetraenoic acid (15-HETE), 18-Hydroxyeicosapentaenoic acid (18-HEPE) or 17-Hydroxydocosahexaenoic acid (17-HDHA). In addition, the influence of 5-LO activating protein (FLAP) inhibition on SPM formation in PMNL was assessed.Intact human PMNL preferred ARA over DHA for lipid mediator formation. In contrast, in incubations supplemented with the SPM precursor lipids DHA-derived 17-HDHA was preferred over 15-HETE and 18-HEPE. SPM formation in the cells was dominated by 5(S),15(S)-diHETE (800 pmol/20 mio cells) and Resolvin D5 (2300 pmol/20 mio cells). Formation of lipoxins (<10 pmol/20 mio cells), E-series (<70 pmol/20 mio cells) and other D-series resolvins (<20 pmol/20 mio cells) was low and only detected after addition of the precursor lipids. Upon destruction of cell integrity, formation of lipoxins and 5(S),15(S)-diHETE increased while formation of 17-HDHA- and 18-HEPE-derived SPMs was attenuated. Recombinant 5-LO did not accept the precursors for SPM formation and FLAP inhibition prevented the formation of the 5-LO-dependent SPMs. Together with the data on FLAP inhibition our results point to unknown factors that control SPM formation in human neutrophils and also render lipoxin and 5(S),15(S)-diHETE formation independent of membrane association and FLAP when cellular integrity is destroyed.

In Vitro Biosynthetic Pathway Investigations of Neuroprotectin D1 (NPD1) and Protectin DX (PDX) by Human 12-Lipoxygenase, 15-Lipoxygenase-1, and 15-Lipoxygenase-2.

In this paper, human platelet 12-lipoxygenase [h12-LOX (ALOX12)], human reticulocyte 15-lipoxygenase-1 [h15-LOX-1 (ALOX15)], and human epithelial 15-lipoxygenase-2 [h15-LOX-2 (ALOX15B)] were observed to react with docosahexaenoic acid (DHA) and produce 17S-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17S-HpDHA). The kcat/KM values with DHA for h12-LOX, h15-LOX-1, and h15-LOX-2 were 12, 0.35, and 0.43 s-1 μM-1, respectively, which demonstrate h12-LOX as the most efficient of the three. These values are comparable to their counterpart kcat/KM values with arachidonic acid (AA), 14, 0.98, and 0.24 s-1 μM-1, respectively. Comparison of their product profiles with DHA demonstrates that the three LOX isozymes produce 11S-HpDHA, 14S-HpDHA, and 17S-HpDHA, to varying degrees, with 17S-HpDHA being the majority product only for the 15-LOX isozymes. The effective kcat/KM values (kcat/KM × percent product formation) for 17S-HpDHA of the three isozymes indicate that the in vitro value of h12-LOX was 2.8-fold greater than that of h15-LOX-1 and 1.3-fold greater than that of h15-LOX-2. 17S-HpDHA was an effective substrate for h12-LOX and h15-LOX-1, with four products being observed under reducing conditions: protectin DX (PDX), 16S,17S-epoxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid (16S,17S-epoxyDHA), the key intermediate in neuroprotection D1 biosynthesis [NPD1, also known as protectin D1 (PD1)], 11,17S-diHDHA, and 16,17S-diHDHA. However, h15-LOX-2 did not react with 17-HpDHA. With respect to their effective kcat/KM values, h12-LOX was markedly less effective than h15-LOX-1 in reacting with 17S-HpDHA, with a 55-fold lower effective kcat/KM in producing 16S,17S-epoxyDHA and a 27-fold lower effective kcat/KM in generating PDX. This is the first direct demonstration of h15-LOX-1 catalyzing this reaction and reveals an in vitro pathway for PDX and NPD1 intermediate biosynthesis. In addition, epoxide formation from 17S-HpDHA and h15-LOX-1 was negatively affected via allosteric regulation by 17S-HpDHA (Kd = 5.9 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (Kd = 2.5 μM), and 17S-hydroxy-13Z,15E,19Z-docosatrienoic acid (17S-HDTA) (Kd = 1.4 μM), suggesting a possible regulatory pathway in reducing epoxide formation. Finally, 17S-HpDHA and PDX inhibited platelet aggregation, with EC50 values of approximately 1 and 3 μM, respectively. The in vitro results presented here may help advise in vivo PDX and NPD1 intermediate (i.e., 16S,17S-epoxyDHA) biosynthetic investigations and support the benefits of DHA rich diets.

Synthesis and molecular targets of N-13-hydroxy-octadienoyl-ethanolamine, a novel endogenous bioactive 15-lipoxygenase-derived metabolite of N-linoleoyl-ethanolamine found in the skin and saliva

N-Arachidonoyl-ethanolamine (AEA) is an endocannabinoid (eCB) and endogenous lipid mimicking many of the effects of Δ9-tetrahydrocannabinol, notably on brain functions, appetite, pain and inflammation. The eCBs and eCB-like compounds contain fatty acids, the main classes being the monoacylglycerols and the N-acyl-ethanolamines (NAEs). Thus, each long chain fatty acid likely exists under the form of a monoacylglycerol and NAE, as it is the case for arachidonic acid (AA) and linoleic acid (LA). Following their biosynthesis, AA and AEA can be further metabolized into additional eicosanoids, notably by the 15-lipoxygenase pathway. Thus, we postulated that NAEs possessing a 1Z,4Z-pentadiene motif, near their omega end, would be transformed into their 15-lipoxygenase metabolites. As a proof of concept, we investigated N-linoleoyl-ethanolamine (LAE). We successfully synthesized LEA and LEA-d4 as well as their 15-lipoxygenase-derived derivatives, namely 13-hydroxy-9Z,11E-octadecadienoyl-N-ethanolamine (13-HODE-EA) and 13-HODE-EA-d4, using Novozyme 435 immobilized on acrylic resin and soybean lipoxygenase respectively. We also show that both human 15-lipoxygenase-1 and -2 can biosynthesize 13-HODE-EA. Co-incubation of LEA and LA with either human 15-lipoxygenase led to the biosynthesis of 13-HODE-EA and 13-HODE in a ratio equal to or greater than 3:1, indicating that LEA is preferred to LA by these enzymes. Finally, we show that 13-HODE-EA is found in human saliva and skin and is a weak although selective TRPV1 agonist. The full biological importance of 13-HODE-EA remains to be explored.

Anti-inflammatory celastrol promotes a switch from leukotriene biosynthesis to formation of specialized pro-resolving lipid mediators

The pentacyclic triterpenoid quinone methide celastrol (CS) from Tripterygium wilfordii Hook. F. effectively ameliorates inflammation with potential as therapeutics for inflammatory diseases. However, the molecular mechanisms underlying the anti-inflammatory and inflammation-resolving features of CS are incompletely understood. Here we demonstrate that CS potently inhibits the activity of human 5-lipoxygenase (5-LOX), the key enzyme in pro-inflammatory leukotriene (LT) formation, in cell-free assays with IC50 = 0.19–0.49 µM. Employing metabololipidomics using ultra-performance liquid chromatography coupled to tandem mass spectrometry in activated human polymorphonuclear leukocytes or M1 macrophages we found that CS (1 µM) potently suppresses 5-LOX-derived products without impairing the formation of lipid mediators (LM) formed by 12-/15-LOXs as well as fatty acid substrate release. Intriguingly, CS induced the generation of 12-/15-LOX-derived LM including the specialized pro-resolving mediator (SPM) resolvin D5 in human M2 macrophages. Finally, intraperitoneal pre-treatment of mice with 10 mg/kg CS strongly impaired zymosan-induced LT formation and simultaneously elevated the levels of SPM and related 12-/15-LOX-derived LM in peritoneal exudates, spleen and plasma in vivo. Conclusively, CS promotes a switch from LT biosynthesis to formation of SPM which may underlie the anti-inflammatory and inflammation-resolving effects of CS, representing an interesting pharmacological strategy for intervention with inflammatory disorders.

Inhibition of Soybean 15-Lipoxygenase and Human 5-Lipoxygenase by Extracts of Leaves, Stem Bark, Phenols and Catechols Isolated From Lithraea caustica (Anacardiaceae).

Lithraea caustica (Molina) Hook. and Arn. (Anacardiaceae), common name Litre, is an evergreen endemic plant used in the Mapuche Chilean folk medicine. The stem juice of L. caustica mixed with Rubus ulmifolius (blackberry) is used to treat cough and the infusion of leaves is used in baths to treat joint inflammations. In this study, the activities of 3-n-alk(en)yl-catechols, obtained from the dichloromethane extract of the epicuticular compounds of fresh leaves (DCME), stem bark petroleum ether extract (PEE), fractions of phenols and phenol-acid compounds obtained from the methanolic extract (methanolic extract) of defatted leaves and aqueous infusion (AE) from fresh leaves, were evaluated as in vitro inhibitors of soybean 15-lipoxygenase (15-sLOX) and human 5-lipoxygenase (5-hLOX), one of the inflammation pathways. The 3-n-alk(en)yl-catechols were characterized by gas chromatography-mass spectrometry and 1D and 2D nuclear magnetic resonance analysis as mixtures of 3-[(10E)-pentadec-10′-en-1-yl]-catechol, 3-[(10Z)-pentadec-10′-en-1-yl]-catechol and 3-n-pentadecylcatechol. In addition, two fractions, obtained from MeOHE, were characterized by liquid chromatography electrospray ionization tandem mass spectrometric as complex mixtures of known acids and phenolic compounds. DCME, MeOHE and ethyl acetate extract (AcOEtE) extracts showed inhibition against 15-sLOX, and the AE of fresh leaves, showed the best inhibition against 5-hLOX. The mixture of 3-n-alk(en)yl-catechols showed inhibition of 15-sLOX and 5-hLOX. The compounds 3-[(10Z)-pentadec-10′-en-1-yl]-catechol (IC50 2.09 µM) and 3-n-pentadecylcatechol (IC50 2.74 µM) showed inhibition against 5-hLOX. The inhibition values obtained for the 3-n-alk(en)yl-catechols are in the range of well-known inhibitors of 5-hLOX. Acetylation of the 3-n-alk(en)yl-catechols blocks the inhibitory activity, indicating that the free catechol function is necessary for the enzyme inhibition. In addition, the fractions of phenols and phenol-acid compounds showed inhibitory activity against 15-sLOX and the AE, showed a good inhibition against 5-hLOX. These results would be in agreement with the use of L. caustica, as an anti-inflammatory in Mapuche ethnomedicine.

Human lipoxygenase isoforms form complex patterns of double and triple oxygenated compounds from eicosapentaenoic acid

Lipoxygenases (ALOX) are lipid peroxidizing enzymes that catalyze the biosynthesis of pro- and anti-inflammatory lipid mediators and have been implicated in (patho-)physiological processes. In humans, six functional ALOX isoforms exist and their arachidonic acid oxygenation products have been characterized. Products include leukotrienes and lipoxins which are involved in the regulation of inflammation and resolution. Oxygenation of n3-polyunsaturated fatty acids gives rise to specialized pro-resolving mediators, e.g. resolvins. However, the catalytic activity of different ALOX isoforms can lead to a multitude of potentially bioactive products. Here, we characterized the patterns of oxygenation products formed by human recombinant ALOX5, ALOX15, ALOX15B and ALOX12 from eicosapentaenoic acid (EPA) and its 18-hydroxy derivative 18-HEPE with particular emphasis on double and triple oxygenation products. ALOX15 and ALOX5 formed a complex mixture of various double oxygenation products from EPA, which include 5,15-diHEPE and various 8,15-diHEPE isomers. Their biosynthetic mechanisms were explored using heavy oxygen isotopes (H218O, 18O2 gas) and three catalytic activities contributed to product formation: i) fatty acid oxygenase activity, ii) leukotriene synthase activity, iii) lipohydroperoxidase activity. For ALOX15B and ALOX12 more specific product patterns were identified, which was also the case when these enzymes reacted in concert with ALOX5. Several double oxygenated compounds were formed from 18-HEPE by ALOX5, ALOX15B and ALOX12 including previously identified resolvins (RvE2, RvE3), while formation of triple oxygenation products, e.g. 5,17,18-triHEPE, required ALOX5. Taken together our data show that EPA can be converted by human ALOX isoforms to a large number of secondary oxygenation products, which might exhibit bioactivity.

Design, Synthesis and Biological Evaluation of Ferrocenyl Thiazole and Thiazolo[5,4-d]thiazole Catechols as Inhibitors of 5-hLOX and as Antibacterials against Staphylococcus aureus. Structural Relationship and Computational Studies

In the search for new human 5-lipoxygenase (5-hLOX) inhibitors that could be used for the treatment of a variety of inflammation-related diseases, three new ferrocenyl complexes derived from 2,4-th...

Ferroptosis inhibitory constituents from the fruits of Cullen corylifolium

In current study, we studied the phytochemicals of Cullen corylifolium (fruits) in which we identify twenty compounds, including two coumarins (1 and 2), three coumestans (3–5), fourchalcone (6–9), three dihydroflavones (10–12), four isoflavones (13–16), one flavonoid (17) and three meroterpenes (18–20). Among these, compounds 4, 5 and 12 were isolated from C. corylifolium for the first time. The ferroptosis inhibitory effects of the isolated phytochemicals were assessed using erastin-exposed HT22 mouse hippocampal cells. Compounds 3 and 18 showed the most potent inhibition with the IC50 values of 5.21 μM and 5.41 μM, respectively. Moreover, molecular docking study showed that compound 3 possessed tremendous inhibitory affinity for human 5-lipoxygenase (5-LOX) and Kelch-like ECH-related protein 1: nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) protein-protein interactions, two important ferroptosis-related targets. These findings indicate that compound 3 (psoralidin) may be a potential therapeutic agent for the treatment of ferroptosis-related diseases.

Human eosinophils and neutrophils biosynthesize novel 15-lipoxygenase metabolites from 1-linoleoyl-glycerol and N-linoleoyl-ethanolamine

Abstract BACKGROUND The endocannabinoids 2-AG and AEA are lipids regulating many physiological processes, notably inflammation. The endocannabinoidome includes other monoacylglycerols (MAG) and N-acyl-ethanolamines (NAE) such as 1-linoleoyl-glycerol (1-LG) and N-linoleoyl-ethanolamine (LEA). Endocannabinoid hydrolysis inhibitors are now being tested as potential anti-inflammatory agents. By increasing MAG and/or NAE levels, these inhibitors will likely increase the levels of their metabolites. Herein we investigated whether 1-LG and LEA were substrates for the 15-lipoxygenase pathway, which is strongly involved in asthma and its severity. We thus assessed how human eosinophils and neutrophils biosynthesized the 15-lipoxygenase metabolites of 1-LG and LEA. Linoleic acid (LA), a well-documented substrate of 15-lipoxygenases, was used as positive control. RESULTS We synthesized the putative 15-lipoxygenase metabolites of 1-LG and LEA using Novozym435 and soybean lipoxygenase. Eosinophils, which express the 15-lipoxygenase-1, metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was almost complete after 5 minutes. Substrate preference of eosinophils was LA&amp;gt;LEA&amp;gt;1-LG. Human neutrophils, which express the 15-lipoxygenase-2, also metabolized LA, 1-LG, and LEA into their 13-hydroxy derivatives. This was maximal after 30 seconds. Substrate preference was LA≫1-LG&amp;gt;LEA. Importantly, the new 15-lipoxygenase metabolites we disclose were found in tissues from humans and mice. CONCLUSIONS We successfully showed that human eosinophils and neutrophils transforms 1-LG and LEA into novel 15-lipoxygenase metabolites. How these new metabolites modulate the inflammatory cascade is now being explored.

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

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 kcat/KM value (14.0 ± 0.8 s-1 μM-1) than that of h15-LOX-1 (0.36 ± 0.08 s-1 μM-1) 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 kcat/KM value, 0.0024 ± 0.0002 and 0.11 ± 0.006 s-1 μM-1, respectively. This is the first evidence of h15-LOX-1 to catalyze this reaction and reveals a novel in vitro 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 (Kd = 6.0 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (Kd = 3.5 μM), and 14S-hydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic acid (14S-HDPAω-3) (Kd = 4.0 μM) were shown to decrease 13S,14S-epoxy-DHA production. h12-LOX was also shown to be allosterically regulated by 14S-HpDHA (Kd = 3.5 μM) and 14S-HDPAω-3 (Kd = 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 in vitro biosynthetic pathways may help guide in vivo maresin biosynthetic investigations and possibly direct therapeutic interventions.

12-LOX catalyzes the oxidation of 2-arachidonoyl-lysolipids in platelets generating eicosanoid-lysolipids that are attenuated by iPLA2γ knockout

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

Study of two isoforms of lipoxygenase by kinetic assays, docking and molecular dynamics of a specialised metabolite isolated from the aerial portion of Lithrea caustica (Anacardiaceae) and its synthetic analogs

Our investigation focused on the characterization and study of epicuticular leaf extracts (dichloromethane extract) and certain derivatives of Lithrea caustica (Molina) Hook and Arn. (Anacardiaceae) as inhibitors of 15 soybean and 5 human lipoxygenases (15-sLOX and 5-hLOX). From the epicuticular extract of leaves, the compound (Z)-3-(pentadec-10′-enyl)-catechol (Litreol) was isolated, and three hemisynthetic derivatives were prepared, as they are 3-pentadecylcatechol, (Z)-1,2-diacetyl-3-(pentadec-10′-enyl)-benzene and 1,2-diacetyl-3-pentadecylbenzene. The inhibitory activities for the four compounds against 15-sLOX and 5-hLOX were determined, being (Z)-3-(pentadec-10′-enyl)-catechol (IC50 54.77 μM and 2.09 μM, respectively) and 3-pentadecylcatechol (IC50 55.28 μM and 2.74 μM, respectively), the most interesting compounds assayed. The kinetic studies for (Z)-3-(pentadec-10′-enyl)-catechol and 3-pentadecylcatechol showed a mixed inhibition mechanism to 5-LOX. Finally, docking and molecular dynamics studies were performed to characterize and describe how the chemical structures could be correlated to the decreased 5-hLOX activity observed in the in vitro studies.

Specific overexpression of 15-lipoxygenase in endothelial cells promotes cancer cell death in an in vivo Lewis lung carcinoma mouse model

PurposeLipoxygenases (LOX) have been implicated in carcinogenesis, however both pro- and anti-carcinogenic effects have been reported in different cancer models. Using transgenic mice, which specifically overexpress human 15-lipoxygenase (ALOX15) in endothelial cells (EC), we previously demonstrated significant inhibition of tumor development. In the Lewis lung carcinoma (LLC) model, the primary tumor developed similarly in both wild type (WT) and ALOX15 overexpressing mice. However, metastases development was significantly inhibited in the transgenic mice. Here, we explored the molecular basis for the anti-metastatic effect of endothelial cell specific ALOX15 overexpression. Materials/methodsWe used ALOX15 overexpressing mice, and in-vitro cell model to evaluate the molecular effect of ALOX15 on EC and LLC cells. ResultsWhen LLC cells were injected in WT and ALOX15 overexpressing mice, we observed a higher degree of apoptosis and necrosis in primary and metastatic tumors of ALOX15 overexpressing animals. These anti-carcinogenic and anti-metastatic effects were paralleled by augmented expression of cyclin-dependent kinase inhibitor 1A (CDKN1A; p21) and of the peroxisome proliferators-activated receptor (PPAR)γ and by downregulation of the steady state concentrations of connexin26 mRNA. Consistent with these in vivo effects, ALOX15 overexpression in LLC and HeLa cancer cells in vitro significantly reduced cell viability in culture. In contrast, similar treatment of non-cancerous B2B epithelial cells did not impact cell viability. ConclusionsTaken together, our data suggests that endothelial cell specific overexpression of ALOX15 promotes apoptosis and necrosis in primary and metastatic tumors in mice, by upregulation of P21 and PPARγ expression in adjacent cancer cells.

Synthesis and structure-activity relationships of novel 5-(hydroxamic acid)methyl oxazolidinone derivatives as 5-lipoxygenase inhibitors

Oxazolidinone hydroxamic acid derivatives were synthesised and evaluated for inhibitory activity against leukotriene (LT) biosynthesis in three in vitro cell-based test systems and on direct inhibition of recombinant human 5-lipoxygenase (5-LO). Thirteen of the 19 compounds synthesised were considered active ((50% inhibitory concentration (IC50) ≤ 10 µM in two or more test systems)). Increasing alkyl chain length on the hydroxamic acid moiety enhanced activity and morpholinyl-containing derivatives were more active than N-acetyl-piperizinyl derivatives. The IC50 values in cell-based assay systems were comparable to those obtained by direct inhibition of 5-LO activity, confirming that the compounds are direct inhibitors of 5-LO. Particularly, compounds PH-249 and PH-251 had outstanding potencies (IC50 < 1 µM), comparable to that of the prototype 5-LO inhibitor, zileuton. Pronounced in vivo activity was demonstrated in zymosan-induced peritonitis in mice. These novel oxazolidinone hydroxamic acid derivatives are, therefore, potent 5-LO inhibitors with potential application as anti-allergic and anti-inflammatory agents.

Myxochelin- and Pseudochelin-Derived Lipoxygenase Inhibitors from a Genetically Engineered Myxococcus xanthus Strain

Precursor-directed biosynthesis was used to introduce selected aryl carboxylic acids into the pseudochelin pathway, which had recently been assembled in Myxococcus xanthus. Overall, 14 previously undescribed analogues of the natural products myxochelin B and pseudochelin A were generated and structurally characterized. A subset of 10 derivatives together with their parental molecules were evaluated for their activity toward human 5-lipoxygenase. This testing revealed pseudochelin A as the most potent 5-lipoxygenase inhibitor among the naturally occurring compounds, whereas myxochelin A is the least active. Replacement of the catechol moieties in myxochelin B and pseudochelin A affected the bioactivity to different degrees.

The standardized herbal combination BNO 2103 contained in Canephron® N alleviates inflammatory pain in experimental cystitis and prostatitis

BackgroundUrinary tract infections are among the most common types of infections and give rise to inflammation with pain as one of the main symptoms. The herbal medicinal product Canephron® N contains BNO 2103, a defined mixture of pulverized rosemary leaves, centaury herb, and lovage root, and has been used in the treatment of urinary tract infections for more than 25 years. PurposeTo test the hypothesis that BNO 2103 reduces pain in cystitis and prostatitis by virtue of anti-inflammatory properties, and to reveal potential mechanisms underlying the anti-inflammatory features. Study designBNO 2103 was studied for anti-inflammatory and analgesic properties in three animal models in vivo, and the mode of action underlying the anti-inflammatory features was investigated in human leukocytes and cell-free assays in vitro. MethodsTo assess the anti-inflammatory and analgesic efficacy of BNO 2103 we employed cyclophosphamide-induced cystitis and carrageenan-induced prostatitis in rats, and zymosan-induced peritonitis in mice. Human neutrophils and monocytes as well as isolated human 5-lipoxygenase and microsomal prostaglandin E2 synthase-1-containing microsomes were utilized to assess inhibition of leukotriene and/or prostaglandin E2 production by HPLC and/or ELISA. ResultsWhen given orally, BNO 2103 reduced inflammation and hyperalgesia in experimental cystitis in rats, while individual components of BNO 2103 also reduced hyperalgesia. Furthermore, BNO 2103 reduced hyperalgesia in rats with carrageenan-induced prostatitis. Cell-based and cell-free studies implicate inhibition of prostaglandin E2 and leukotriene B4 biosynthesis as potential mechanisms underlying the analgesic and anti-inflammatory effects. ConclusionOur data support the hypothesis that BNO 2103 reduces pain by virtue of its anti-inflammatory properties, possibly related to suppression of prostaglandin E2 and leukotriene B4 formation, and suggest that this combination has the potential to treat clinical symptoms such as inflammatory pain. Thus BNO 2103 may represent an alternative to reduce the use of antibiotics in urinary tract infections.