Identification of 2,4-Dinitro-Biphenyl-Based Compounds as MAPEG Inhibitors.

We identified 2,4-dinitro-biphenyl-based compounds as new inhibitors of leukotriene C4 synthase (LTC4 S) and 5-lipoxygenase-activating protein (FLAP), both members of the "Membrane Associated Proteins in Eicosanoid and Glutathione metabolism" (MAPEG) family involved in the biosynthesis of pro-inflammatory eicosanoids. By molecular docking we evaluated the putative binding against the targets of interest, and by applying cell-free and cell-based assays we assessed the inhibition of LTC4 S and FLAP by the small molecules at low micromolar concentrations. The present results integrate the previously observed inhibitory profile of the tested compounds against another MAPEG member, i. e., microsomal prostaglandin E2 synthase (mPGES)-1, suggesting that the 2,4-dinitro-biphenyl scaffold is a suitable molecular platform for a multitargeting approach to modulate pro-inflammatory mediators in inflammation and cancer treatment.

Modulation of microRNA processing by 5-lipoxygenase.

The miRNA biogenesis is tightly regulated to avoid dysfunction and consequent disease development. Here, we describe modulation of miRNA processing as a novel noncanonical function of the 5-lipoxygenase (5-LO) enzyme in monocytic cells. In differentiated Mono Mac 6 (MM6) cells, we found an in situ interaction of 5-LO with Dicer, a key enzyme in miRNA biogenesis. RNA sequencing of small noncoding RNAs revealed a functional impact, knockout of 5-LO altered the expression profile of several miRNAs. Effects of 5-LO could be observed at two levels. qPCR analyses thus indicated that (a) 5-LO promotes the transcription of the evolutionarily conserved miR-99b/let-7e/miR-125a cluster and (b) the 5-LO-Dicer interaction downregulates the processing of pre-let-7e, resulting in an increase in miR-125a and miR-99b levels by 5-LO without concomitant changes in let-7e levels in differentiated MM6 cells. Our observations suggest that 5-LO regulates the miRNA profile by modulating the Dicer-mediated processing of distinct pre-miRNAs. 5-LO inhibits the formation of let-7e which is a well-known inducer of cell differentiation, but promotes the generation of miR-99b and miR-125a known to induce cell proliferation and the maintenance of leukemic stem cell functions.

12-Oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG10), a novel glutathione-containing eicosanoid generated via the 12-lipoxygenase pathway in human platelets.

Biologically active glutathione (GSH) conjugates of oxygenated fatty acids comprise a group of pro- and anti-inflammatory lipid mediators. While arachidonic acid (AA)-derived conjugates, as the cysteinyl leukotrienes (cys-LTs) and eoxins (EXs) have pro-inflammatory properties, conjugates in tissue regeneration (CTRs) biosynthesized from docosahexaenoic acid (DHA) exhibit pro-resolving activity. Human platelets express abundant amounts of platelet-type 12-lipoxygenase (pt12-LOX) and leukotriene C4 synthase (LTC4S). However, the only two described GSH conjugates formed by platelets are the AA-derived cys-LTs and the recently reported maresin CTRs (MCTRs). While cys-LTs are biosynthesized in a transcellular mechanism via the action of 5-LOX and LTC4S, MCTR1 is formed by 12-LOX and a yet unidentified GSH S-transferase (GST). Here, we present a novel GSH conjugate formed from AA via the 12-LOX pathway in human platelets. The 12-oxo-glutathione adduct, 12-oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG10), was identified by mass spectrometry using positive electrospray ionization. The structural proposal is supported by fragmentation data of the labeled metabolite obtained after incubation of deuterated AA (AA-d8). In platelets as well as in HEK293 cells stably expressing pt12-LOX, TOG10 biosynthesis was inhibited by the 12-LOX inhibitor ML-355 (5 µM), which confirms the involvement of pt12-LOX. Interestingly, TOG10 was formed independently of LTC4S in platelets. This is in accordance with the observation that the conjugate was also generated by AA-stimulated HEK_12-LOX cells in absence of LTC4S. Nevertheless, TOG10 can also be formed by LTC4S as the biosynthesis in HEK_12-LOX_LTC4S cells was reduced by the specific LTC4S inhibitor TK04a. In summary, TOG10 was identified as a new AA-derived GSH conjugate generated in human platelets via the action of pt12-LOX in combination with a GST.

Finding New Molecular Targets of Familiar Natural Products Using In Silico Target Prediction.

Natural products comprise a rich reservoir for innovative drug leads and are a constant source of bioactive compounds. To find pharmacological targets for new or already known natural products using modern computer-aided methods is a current endeavor in drug discovery. Nature's treasures, however, could be used more effectively. Yet, reliable pipelines for the large-scale target prediction of natural products are still rare. We developed an in silico workflow consisting of four independent, stand-alone target prediction tools and evaluated its performance on dihydrochalcones (DHCs)-a well-known class of natural products. Thereby, we revealed four previously unreported protein targets for DHCs, namely 5-lipoxygenase, cyclooxygenase-1, 17ß-hydroxysteroid dehydrogenase 3, and aldo-keto reductase 1C3. Moreover, we provide a thorough strategy on how to perform computational target predictions and guidance on using the respective tools.

Liquid chromatography-coupled mass spectrometry analysis of glutathione conjugates of oxygenated polyunsaturated fatty acids.

Glutathione (GSH) conjugates of oxygenated polyunsaturated fatty acids comprise a group of pro-inflammatory and pro-resolving lipid mediators formed in immunocompetent cells. While the pro-inflammatory conjugates such as the cysteinyl leukotrienes (cys-LTs), eoxins (EXs) and five-oxo-GSH conjugate (FOG7) derive from arachidonic acid (AA), the group of conjugates in tissue regeneration (CTRs) such as maresin CTRs (MCTRs), protectin CTRs (PCTRs) and resolvin CTRs (RCTRs) are biosynthesized from docosahexaenoic acid (DHA). Here, we present a gradient UPLC-MS/MS method for the analysis of pro-inflammatory and pro-resolving GSH conjugates using positive electrospray ionization (ESI(+)) and collision-induced fragmentation for unambiguous identification and structural information, and a negative ionization (ESI(-)) mode for quantification of the GSH conjugates. The method was employed to detect GSH conjugates in human platelets and macrophages. MCTRs were detected in platelets upon addition of exogenous docosahexaenoic acid (DHA) and the biosynthesis was independent on leukotriene C4 (LTC4) synthase activity. Pathogenic bacteria stimulated the formation of EXs and PCTRs in M2 macrophages, whereas Ca2+-ionophore activated the biosynthesis of LTC4 in M1 and M2 macrophage phenotypes. Together, our methodology covers the qualitative and quantitative analysis of GSH conjugates and gives an analytical basis for the detection and structural elucidation of cysteinyl-containing lipid mediators.

Impact of food polyphenols on oxylipin biosynthesis in human neutrophils.

The intake of food polyphenols is associated with beneficial impacts on health. Besides anti-oxidative effects, anti-inflammatory properties have been suggested as molecular modes of action, which may result from modulations of the arachidonic acid (AA) cascade. Here, we investigated the effects of a library of food polyphenols on 5-lipoxygenase (5-LOX) activity in a cell-free assay, and in human neutrophils. Resveratrol, its dimer (ε-viniferin), and its imine analogue (IRA) potently blocked the 5-LOX-mediated LT formation in neutrophils with IC50 values in low µM-range. Among the tested flavonoids only the isoflavone genistein showed potent 5-LOX inhibition in neutrophils (IC50 = 0.4 ±â€¯0.1 µM), however was ineffective on isolated 5-LOX. We exclude an interference with the 5-LOX-activating protein (FLAP) in HEK_5-LOX/±FLAP cells and suggest global effects on intact immune cells. Using LC-MS based targeted oxylipin metabolomics, we analyzed the effects of 5-LOX-inhibiting polyphenols on all branches of the AA cascade in Ca2+-ionophore-challenged neutrophils. While ε-viniferin causes a clear substrate shunt towards the remaining AA cascade enzymes (15-LOX, cyclooxygenase - COX-1/2, cytochrome P450), resveratrol inhibited the COX-1/2 pathway and showed a weak attenuation of 12/15-LOX activity. IRA had no impact on 15-LOX activity, but elevated the formation of COX-derived prostaglandins, having no inhibitory effects on COX-1/2. Overall, we show that food polyphenols have the ability to block 5-LOX activity and the oxylipin pattern is modulated with a remarkable compound/structural specificity. Taken the importance of polyphenols for a healthy diet and their concentration in food supplements into account, this finding justifies further investigation.

Targeting biosynthetic networks of the proinflammatory and proresolving lipid metabolome.

Nonsteroidal anti-inflammatory drugs interfere with the metabolism of arachidonic acid to proinflammatory prostaglandins and leukotrienes by targeting cyclooxygenases (COXs), 5-lipoxygenase (LOX), or the 5-LOX-activating protein (FLAP). These and related enzymes act in conjunction with marked crosstalk within a complex lipid mediator (LM) network where also specialized proresolving LMs (SPMs) are formed. Here, we present how prominent LM pathways can be differentially modulated in human proinflammatory M1 and proresolving M2 macrophage phenotypes that, upon exposure to Escherichia coli, produce either abundant prostaglandins and leukotrienes (M1) or SPMs (M2). Targeted liquid chromatography-tandem mass spectrometry-based metabololipidomics was applied to analyze and quantify the specific LM profiles. Besides expected on-target actions, we found that: 1) COX or 15-LOX-1 inhibitors elevate inflammatory leukotriene levels, 2) FLAP and 5-LOX inhibitors reduce leukotrienes in M1 but less so in M2 macrophages, 3) zileuton blocks resolution-initiating SPM biosynthesis, whereas FLAP inhibition increases SPM levels, and 4) that the 15-LOX-1 inhibitor 3887 suppresses SPM formation in M2 macrophages. Conclusively, interference with discrete LM biosynthetic enzymes in different macrophage phenotypes considerably affects the LM metabolomes with potential consequences for inflammation-resolution pharmacotherapy. Our data may allow better appraisal of the therapeutic potential of these drugs to intervene with inflammatory disorders.-Werner, M., Jordan, P. M., Romp, E., Czapka, A., Rao, Z., Kretzer, C., Koeberle, A., Garscha, U., Pace, S., Claesson, H.-E., Serhan, C. N., Werz, O., Gerstmeier, J. Targeting biosynthetic networks of the proinflammatory and proresolving lipid metabolome.

A Multi-step Virtual Screening Protocol for the Identification of Novel Non-acidic Microsomal Prostaglandin†E2 Synthase-1 (mPGES-1) Inhibitors.

Microsomal prostaglandin E2 synthase-1 (mPGES-1) is a potential therapeutic target for the treatment of inflammatory diseases and certain types of cancer. To identify novel scaffolds for mPGES-1 inhibition, we applied a virtual screening (VS) protocol that comprises molecular docking, fingerprints-based clustering with diversity-based selection, protein-ligand interactions fingerprints, and molecular dynamics (MD) simulations with molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations. The hits identified were carefully analyzed to ensure the selection of novel scaffolds that establish stable interactions with key residues in the mPGES-1 binding pocket and inhibit the catalytic activity of the enzyme. As a result, we discovered two promising chemotypes, 4-(2-chlorophenyl)-N-[(2-{[(propan-2-yl)sulfamoyl]methyl}phenyl)methyl]piperazine-1-carboxamide (6) and N-(4-methoxy-3-{[4-(6-methyl-1,3-benzothiazol-2-yl)phenyl]sulfamoyl}phenyl)acetamide (8), as non-acidic mPGES-1 inhibitors with IC50 values of 1.2 and 1.3 µm, respectively. Minimal structural optimization of 8 resulted in three more compounds with promising improvements in inhibitory activity (IC50 : 0.3-0.6 µm). The unprecedented chemical structures of 6 and 8, which are amenable to further derivatization, reveal a new and attractive approach for the development of mPGES-1 inhibitors with potential anti-inflammatory and anticancer properties.

Melleolides from Honey Mushroom Inhibit 5-Lipoxygenase via Cys159.

5-Lipoxygenase (5-LO) initiates the biosynthesis of pro-inflammatory leukotrienes from arachidonic acid, which requires the nuclear membrane-bound 5-LO-activating protein (FLAP) for substrate transfer. Here, we identified human 5-LO as a molecular target of melleolides from honey mushroom (Armillaria mellea). Melleolides inhibit 5-LO via an α,ß-unsaturated aldehyde serving as Michael acceptor for surface cysteines at the substrate entrance that are revealed as molecular determinants for 5-LO activity. Experiments with 5-LO mutants, where select cysteines had been replaced by serine, indicated that the investigated melleolides suppress 5-LO product formation via two distinct modes of action: (1) by direct interference with 5-LO activity involving two or more of the cysteines 159, 300, 416, and 418, and (2) by preventing 5-LO/FLAP assemblies involving selectively Cys159 in 5-LO. Interestingly, replacement of Cys159 by serine prevented 5-LO/FLAP assemblies as well, implying Cys159 as determinant for 5-LO/FLAP complex formation at the nuclear membrane required for leukotriene biosynthesis.

Pharmacological profile and efficiency in vivo of diflapolin, the first dual inhibitor of 5-lipoxygenase-activating protein and soluble epoxide hydrolase.

Arachidonic acid (AA) is metabolized to diverse bioactive lipid mediators. Whereas the 5-lipoxygenase-activating protein (FLAP) facilitates AA conversion by 5-lipoxygenase (5-LOX) to pro-inflammatory leukotrienes (LTs), the soluble epoxide hydrolase (sEH) degrades anti-inflammatory epoxyeicosatrienoic acids (EETs). Accordingly, dual FLAP/sEH inhibition might be advantageous drugs for intervention of inflammation. We present the in vivo pharmacological profile and efficiency of N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N'-(3,4-dichlorophenyl)urea (diflapolin) that dually targets FLAP and sEH. Diflapolin inhibited 5-LOX product formation in intact human monocytes and neutrophils with IC50 = 30 and 170 nM, respectively, and suppressed the activity of isolated sEH (IC50 = 20 nM). Characteristic for FLAP inhibitors, diflapolin (I) failed to inhibit isolated 5-LOX, (II) blocked 5-LOX product formation in HEK cells only when 5-LOX/FLAP was co-expressed, (III) lost potency in intact cells when exogenous AA was supplied, and (IV) prevented 5-LOX/FLAP complex assembly in leukocytes. Diflapolin showed target specificity, as other enzymes related to AA metabolism (i.e., COX1/2, 12/15-LOX, LTA4H, LTC4S, mPGES1, and cPLA2) were not inhibited. In the zymosan-induced mouse peritonitis model, diflapolin impaired vascular permeability, inhibited cysteinyl-LTs and LTB4 formation, and suppressed neutrophil infiltration. Diflapolin is a highly active dual FLAP/sEH inhibitor in vitro and in vivo with target specificity to treat inflammation-related diseases.

Development of smart cell-free and cell-based assay systems for investigation of leukotriene C4 synthase activity and evaluation of inhibitors.

Cysteinyl leukotrienes (cys-LTs) cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) yielding the unstable leukotriene A4 (LTA4) that is subsequently conjugated with glutathione (GSH) by LTC4 synthase (LTC4S). Cys-LT receptor antagonists and LTC4S inhibitors have been developed, but only the former have reached the market. High structural homology to related enzymes and lack of convenient test systems due to instability of added LTA4 have hampered the development of LTC4S inhibitors. We present smart cell-free and cell-based assay systems based on in situ-generated LTA4 that allow studying LTC4S activity and investigating LTC4S inhibitors. Co-incubations of microsomes from HEK293 cells expressing LTC4S with isolated 5-LOX efficiently converted exogenous AA to LTC4 (~1.3µg/200µg protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTC4S with Ca2+-ionophore A23187 and 20µM AA resulted in strong LTC4 formation (~250ng/106 cells). MK-886, a well-known 5-LOX activating protein (FLAP) inhibitor that also acts on LTC4S, consistently inhibited LTC4 formation in all assay types (IC50=3.1-3.5µM) and we successfully confirmed TK04a as potent LTC4S inhibitor in these assay systems (IC50=17 and 300nM, respectively). We demonstrated transcellular LTC4 biosynthesis between neutrophils or 5-LOX-expressing HEK293 cells that produce LTA4 from AA and HEK293 cells expressing LTC4S that transform LTA4 to LTC4. In conclusion, our assay approaches are advantageous as the substrate LTA4 is generated in situ and are suitable for studying enzymatic functionality of LTC4S including site-directed mutations and evaluation of LTC4S inhibitors.

Characterization of the interaction of human 5-lipoxygenase with its activating protein FLAP.

Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes (LTs), important mediators of inflammation. Cellular 5-LO activity is regulated in a complex manner, e.g. by calcium influx, the cellular redox status or 5-LO phosphorylation. Being a mobile enzyme, 5-LO migrates from the cytosol to the nuclear envelope where it is believed to interact with 5-lipoxygenase-activating protein (FLAP) and receives the substrate arachidonic acid (AA). 5-LO contains four cysteine residues located close to the AA entry site. In the present study, we show that in vitro glutathionylation of recombinant purified 5-LO wildtype (WT) as well as 5-LO 4C, a mutant where the four surface cysteines are replaced by serines (Cys159/300/416/418Ser), does not alter the product synthesis. However, in 5-LO/FLAP-transfected HeLa cells, treatment with the thiol-oxidizing agent diamide which promotes glutathionylation at surface Cys residues led to a decreased LT synthesis by 5-LO WT. In contrast to the WT enzyme, LT formation of the 4C mutant was stimulated by addition of diamide. Immunofluorescence studies in human monocytes and HEK293 cells, expressing 5-LO and FLAP, revealed that diamide prevented the translocation of 5-LO WT whereas it enhanced the translocation of the fourfold cysteine mutant. Therefore, we could demonstrate that the interface, involving the four cysteines 159, 300, 416 and 418, is important for the translocation to the nuclear membrane and the colocalization with FLAP.

Design, synthesis and evaluation of semi-synthetic triazole-containing caffeic acid analogues as 5-lipoxygenase inhibitors.

In this work the synthesis, structure-activity relationship (SAR) and biological evaluation of a novel series of triazole-containing 5-lipoxygenase (5-LO) inhibitors are described. The use of structure-guided drug design techniques provided compounds that demonstrated excellent 5-LO inhibition with IC50 of 0.2 and 3.2 µm in cell-based and cell-free assays, respectively. Optimization of binding and functional potencies resulted in the identification of compound 13d, which showed an enhanced activity compared to the parent bioactive compound caffeic acid 5 and the clinically approved zileuton 3. Compounds 15 and 16 were identified as lead compounds in inhibiting 5-LO products formation in neutrophils. Their interference with other targets on the arachidonic acid pathway was also assessed. Cytotoxicity tests were performed to exclude a relationship between cytotoxicity and the increased activity observed after structure optimization.

Discovery of novel, non-acidic mPGES-1 inhibitors by virtual screening with a multistep protocol.

Microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors are considered as potential therapeutic agents for the treatment of inflammatory pain and certain types of cancer. So far, several series of acidic as well as non-acidic inhibitors of mPGES-1 have been discovered. Acidic inhibitors, however, may have issues, such as loss of potency in human whole blood and in vivo, stressing the importance of the design and identification of novel, non-acidic chemical scaffolds of mPGES-1 inhibitors. Using a multistep virtual screening protocol, the Vitas-M compound library (∼1.3 million entries) was filtered and 16 predicted compounds were experimentally evaluated in a biological assay in vitro. This approach yielded two molecules active in the low micromolar range (IC50 values: 4.5 and 3.8 µM, respectively).

Effects of PCB126 and 17ß-oestradiol on endothelium-derived vasoactive factors in human endothelial cells.

Epidemiological and experimental studies suggest an association between elevated serum levels of co-planar PCBs and hypertension, and one study indicate that this effect is dependent on the level of oestrogen. This study investigated the effects of 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 17ß-oestradiol (E2) on vasoactive factors in human umbilical vein endothelial cells (HUVEC). The results reveal that PCB126 stimulated the vasoconstriction factors COX-2 and PGF(2α) in HUVEC. An up-regulation of COX-2 expression was demonstrated using qRT-PCR, western blot and immunofluorescence and increased production of PGF(2α) was demonstrated using LC/MS² and enzyme immunoassay. Also, PCB126 slightly increased ROS production and decreased NO production in HUVEC. The addition of E2 enhanced PCB126-induced transcription of CYP1A1, CYP1B1 and COX-2 in HUVEC whereas an increased transcription of eNOS only occurred following combined treatment with E2 and PCB126. Immunofluorescence demonstrated that HUVEC expressed AHR and ERß but lacked ERα and the involvement of AHR and ERß on the effects of PCB126 was examined by the addition of AHR and ER antagonists. The binding of PCB126 to AHR was critical for the effects of PCB126 whereas the role of ERß was equivocal. In conclusion, these studies suggest that PCB126 induced changes in human endothelial cells that are characteristic for endothelial dysfunction in human hypertension and that PCB126-induced transcription of genes important for vascular function in human endothelial cells can be elevated by increased oestrogen levels. These findings may help understanding the mechanism for the association between PCB126 exposure and hypertension reported in human subjects and experimental animals.

Reaction mechanism of 5,8-linoleate diol synthase, 10R-dioxygenase, and 8,11-hydroperoxide isomerase of Aspergillus clavatus.

Aspergilli express fusion proteins of an animal haem peroxidase domain with fatty acid dioxygenase (DOX) activity ( approximately 600 amino acids) and a functional or non-functional hydroperoxide isomerase/cytochrome P450 domain ( approximately 500 amino acids with EXXR and GPHXCLG motifs). 5,8-Linoleate diol synthases (LDS; ppoA) and 10R-DOX (ppoC) of Aspergillusnidulans and A. fumigatus belong to this group. Our objective was to determine the oxylipins formed from linoleic acid by A. clavatus and their mechanism of biosynthesis. A. clavatus oxidized linoleic acid to (8R)-hydroperoxylinoleic acid (8R-HPODE), (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE), and to (5S,8R)-dihydroxy- and (8R,11S)-dihydroxylinoleic acids (DiHODE) as major products. This occurred by abstraction of the pro-S hydrogen at C-8 and antarafacial dioxygenation at C-8 or at C-10 with double bond migration. 8R-HPODE was then isomerized to 5S,8R-DiHODE and to 8R,11S-DiHODE by abstraction of the pro-S hydrogens at C-5 and C-11 of 8R-HPODE, respectively, followed by suprafacial oxygenation. The genome of A. clavatus codes for two enzymes, which can be aligned with >65% amino acid identity to 10R-DOX and 5,8-LDS, respectively. The 5,8-LDS homologue likely forms and isomerizes 8R-HPODE to 5S,8R-DiHODE. A third gene (ppoB) codes for a protein which carries a serine residue at the cysteine position of the P450 motif. This Cys to Ser replacement is known to abolish P450 2B4 catalysis and the hydroperoxide isomerase activity of 5,8-LDS, suggesting that ppoB of A. clavatus may not be involved in the biosynthesis of 8R,11S-DiHODE.

A lipoxygenase with dual positional specificity is expressed in olives (Olea europaea L.) during ripening.

Plant lipoxygenases (LOXs) are a class of widespread dioxygenases catalysing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological roles remain to be clarified. With the aim to clarify the occurrence of LOXs in olives and their contribution to the elaboration of the olive oil aroma, we cloned and characterized the first cDNA of the LOX isoform which is expressed during olive development. The open reading frame encodes a polypeptide of 864 amino acids. This olive LOX is a type-1 LOX which shows a high degree of identity at the peptide level towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme shows a dual positional specificity, as it forms both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio, and would be defined as 9/13-LOX. Although a LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest that there are at least two Lox genes expressed in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity and its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered.