Specialized pro-resolving mediators (SPMs) have been suggested to act as key factors that mediate the resolution of inflammation. With the availability of synthetic standards, pharmacological studies have garnered support for the hypothesis. However, assessment of the catalytic properties of lipoxygenases as well as investigations of the signalling of the proposed SPM receptors raise serious doubt that SPMs are endogenous mediators of the resolution of inflammation. In line with this, careful evaluation of published data on SPM detection revealed a lack of evidence for many reported SPMs in biological samples due to analytical LC-MS/MS methods that do not meet the quality standards of the scientific community, or they rely on ELISAs that lack selectivity and are not suited for the establishment of the presence of SPMs in complex biological samples. Furthermore, artefacts due to autoxidation of PUFAs can be misinterpreted as SPM formation. In this review, we critically assess the published reports on the biosynthesis and the analysis of SPMs and summarize the current status in the field.

The tumor microenvironment (TME) is a pro-cancerous niche harboring immunosuppressive factors that are secreted by cancer cells and the surrounding cancer-supportive tissue, such as kynurenine, prostaglandin E2 and transforming growth factor β (TGFβ). These factors dampen the activity of cytotoxic lymphocytes like natural killer (NK) cells, allowing evasion of immune cell-mediated killing. To identify small molecules that counteract the immunosuppressive effect of the TME and restore NK cell-mediated cytotoxicity, we developed a phenotypic co-culture assay of cancer cells and primary lymphocytes suitable for medium-throughput screening. We discovered small molecules that restore NK cell-mediated cytotoxicity through diverse mechanisms. The potent TGFβ type I receptor (TGFβR-1) inhibitor, RepSox, stood out as superior to other TGFβR-1 inhibitors due to its ability to abolish the effects of both inhibitory factors used in our setup. This mode of action goes beyond TGFβR-1 inhibition and is related to the simultaneous abrogation of cyclooxygenase 1 (COX1) activity.

Several oxylipins are potent lipid mediators that regulate diverse aspects of health and disease and whose quantitative analysis by liquid chromatography-mass spectrometry (LC-MS) presents substantial technical challenges. As members of the lipidomics community, we developed technical recommendations to ensure best practices when quantifying oxylipins by LC-MS.

The tumor microenvironment (TME) is a pro-cancerous niche harboring immunosuppressive factors that are secreted by cancer cells and the surrounding cancer-supportive tissue, such as kynurenine, prostaglandin E2 and transforming growth factor β (TGFβ). These factors dampen the activity of cytotoxic lymphocytes like natural killer (NK) cells, allowing evasion of immune cell-mediated killing. To identify small molecules that counteract the immunosuppressive effect of the TME and restore NK cell-mediated cytotoxicity, we developed a phenotypic co-culture assay of cancer cells and primary lymphocytes suitable for medium-throughput screening. We discovered small molecules that restore NK cell-mediated cytotoxicity through diverse mechanisms. The potent TGFβ type I receptor (TGFβR-1) inhibitor, RepSox, stood out as superior to other TGFβR-1 inhibitors due to its ability to abolish the effects of both inhibitory factors used in our setup. This mode of action goes beyond TGFβR-1 inhibition and is related to the simultaneous abrogation of cyclooxygenase 1 (COX1) activity. ### Competing Interest Statement The authors have declared no competing interest.

Soluble epoxide hydrolase (sEH) represents a promising target for inflammation-related diseases as it hydrolyzes highly anti-inflammatory epoxy-fatty acids (EpFAs) to the less active corresponding diols.1 sEH harbours two distinct catalytic domains, the C-terminal hydrolase domain and the N-terminal phosphatase domain which are connected by a proline-rich linker. Although potent inhibitors of enzymatic activity are available for both domains, sEH-PROTACs offer the unique ability to simultaneously degrade both domains, mimicking the sEH knockout phenotype associated with beneficial effects as reducing inflammation, attenuating neuroinflammation, and delaying the progression of Alzheimer's disease. Herein, we report the structure-based development of a potent sEH-PROTAC as a useful tool compound for the investigation of sEH. In order to facilitate a rapid testing of the synthesized compounds a cell-based sEH degradation assay was developed based on the HiBiT-technology. A structure-activity-relationship (SAR) investigation was performed, based on the crystal structure of previously published sEH inhibitor FL217 where we identified two possible exit vectors. We designed and synthesized a set of 24 PROTACs with varying linkers in a combinatorial manner. Furthermore, co-crystallization of sEH with two selected PROTACs allowed us to explore the binding mode and rationalize the appropriate linker length. After biological and physicochemical investigation, the most suitable PROTAC 23 was identified and applied to degrade sEH in primary human macrophages, marking the successful translation and applicability to non-artificial systems. ### Competing Interest Statement The authors have declared no competing interest.

5-Lipoxygenase (5-LO), a fatty acid oxygenase, is the central enzyme in leukotriene (LT) biosynthesis, potent arachidonic acid-derived lipid mediators released by innate immune cells, that control inflammatory and allergic responses. In addition, through interaction with 12- and 15-lipoxgenases, the enzyme is involved in the formation of omega-3 fatty acid-based oxylipins, which are thought to be involved in the resolution of inflammation. The expression of 5-LO is frequently deregulated in solid and liquid tumors, and there is strong evidence that the enzyme plays an important role in carcinogenesis. However, global inhibition of LT formation and signaling has not yet shown the desired success in clinical trials. Curiously, the release of 5-LO-derived lipid mediators from tumor cells is often low, and the exact mechanism by which 5-LO influences tumor cell function is poorly understood. Recent data now show that in addition to releasing oxylipins, 5-LO can also influence gene expression in a lipid mediator-independent manner. These non-canonical functions, including modulation of miRNA processing and transcription factor shuttling, most likely influence cancer cell function and the tumor microenvironment and might explain the low clinical efficacy of pharmacological strategies that previously only targeted oxylipin formation and signaling by 5-LO. This review summarizes the canonical and non-canonical functions of 5-LO with a particular focus on tumorigenesis, highlights unresolved issues, and suggests future research directions.

Effect of CJ-13,610 on Wnt-target genes in the PML/RARα-positive cell line NB4.

The leukotriene (LT) pathway is positively correlated with the progression of solid malignancies, but the factors that control the expression of 5-lipoxygenase (5-LO), the central enzyme in LT biosynthesis, in tumors are poorly understood. Here, we report that 5-LO along with other members of the LT pathway is up-regulated in multicellular colon tumor spheroids. This up-regulation was inversely correlated with cell proliferation and activation of PI3K/mTORC-2- and MEK-1/ERK-dependent pathways. Furthermore, we found that E2F1 and its target gene MYBL2 were involved in the repression of 5-LO during cell proliferation. Importantly, we found that this PI3K/mTORC-2- and MEK-1/ERK-dependent suppression of 5-LO is also existent in tumor cells from other origins, suggesting that this mechanism is widely applicable to other tumor entities. Our data show that tumor cells fine-tune 5-LO and LT biosynthesis in response to environmental changes repressing the enzyme during proliferation while making use of the enzyme under cell stress conditions, implying that tumor-derived 5-LO plays a role in the manipulation of the tumor stroma to quickly restore cell proliferation.

Effect of Zileuton on stem cell capacity in PML/RARα positive HSPCs.

Direct interaction of 5-LO with β-catenin.

Supplementary Figure Legends

Specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins are formed by the consecutive action of 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- or 15-lipoxygenases using arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid as substrate. Lipoxins are trihydroxylated oxylipins which are formed from arachidonic and eicosapentaenoic acid. The latter can also be converted to diand trihydroxylated resolvins of the E series, whereas docosahexaenoic acid is the substrate for the formation of di- and trihydroxylated resolvins of the D series. Here, we summarize the formation of lipoxins and resolvins in leukocytes. From the data published so far, it becomes evident that FLAP is required for the biosynthesis of most of the lipoxins and resolvins. Even in the presence of FLAP, formation of the trihydroxylated SPMs (lipoxins, RvD1-RvD4, RvE1) in leukocytes is very low or undetectable which is obviously due to the extremely low epoxide formation by 5-LO from oxylipins such as 15-H(p)ETE, 18-H(p)EPE or 17-H(p)DHA. As a result, only the dihydroxylated oxylipins (5 S,15S-diHETE, 5 S,15S-diHEPE) and resolvins (RvD5, RvE2, RvE4) can be consistently detected using leukocytes as SPM source. However, the reported levels of these dihydroxylated lipid mediators are still much lower than those of the typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g. 5-HETE), leukotrienes or cyclooxygenase-derived prostaglandins. Since 5-LO expression is mainly restricted to leukocytes these cells are considered as the main source of SPMs. The low formation of trihydroxylated SPMs in leukocytes, the fact that they are hardly detected in biological samples as well as the lack of functional signaling by their receptors make it highly questionable that trihydroxylated SPMs play a role as endogenous mediators in the resolution of inflammation.

Laser scan confocal analysis of 5-LO and RARα protein localization in U937-PR9 cells.

Effect of sEH-/- on replating efficiency of PML/RARα-expressing HSPCs.

This figure depicts an additional IF analysis on NB4 cells showing the interactioin between b-catenin and 5-LO upon threatment with CJ13,610

Pharmaceutical inhibition of all three major arachidonic acid pathways and its effect on replating efficiency of PML/RARα-expressing murine HSPCs.

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.

Formation of specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins usually involves arachidonic acid 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- and 15-lipoxygenating paralogues (15-LO1, ALOX15; 15-LO2, ALOX15B; 12-LO, ALOX12). Typically, SPMs are thought to be formed via consecutive steps of oxidation of polyenoic fatty acids such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. One hallmark of SPM formation is that reported levels of these lipid mediators are much lower than typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g., 5-HETE), leukotrienes or certain cyclooxygenase-derived prostaglandins. Thus, reliable detection and quantification of these metabolites is challenging. This paper is aimed at critically evaluating i) the proposed biosynthetic pathways of SPM formation, ii) the current knowledge on SPM receptors and their signaling cascades and iii) the analytical methods used to quantify these pro-resolving mediators in the context of their instability and their low concentrations. Based on current literature it can be concluded that i) there is at most, a low biosynthetic capacity for SPMs in human leukocytes. ii) The identity and the signaling of the proposed G-protein-coupled SPM receptors have not been supported by studies in knock-out mice and remain to be validated. iii) In humans, SPM levels were neither related to dietary supplementation with their ω-3 polyunsaturated fatty acid precursors nor were they formed during the resolution phase of an evoked inflammatory response. iv) The reported low SPM levels cannot be reliably quantified by means of the most commonly reported methodology. Overall, these questions regarding formation, signaling and occurrence of SPMs challenge their role as endogenous mediators of the resolution of inflammation.

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.

5-Lipoxygenase (5-LO), the central enzyme in the biosynthesis of leukotrienes, is frequently expressed in human solid malignancies even though the enzyme is not present in the corresponding healthy tissues. There is little knowledge on the consequences of this expression for the tumor cells regarding gene expression and cellular function. We established a knockout (KO) of 5-LO in different cancer cell lines (HCT-116, HT-29, U-2 OS) and studied the consequences on global gene expression using next generation sequencing. Furthermore, cell viability, proliferation, migration and multicellular tumor spheroid (MCTS) formation were studied in these cells. Our results show that 5-LO influences the gene expression and cancer cell function in a cell type-dependent manner. The enzyme affected genes involved in cell adhesion, extracellular matrix formation, G protein signaling and cytoskeleton organization. Furthermore, absence of 5-LO elevated TGFβ2 expression in HCT-116 cells while MCP-1, fractalkine and platelet-derived growth factor expression was attenuated in U-2 OS cells suggesting that tumor cell-derived 5-LO shapes the tumor microenvironment. In line with the gene expression data, KO of 5-LO had an impact on cell proliferation, motility and MCTS formation. Interestingly, pharmacological inhibition of 5-LO only partly mimicked the KO suggesting that also noncanonical functions are involved.