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Inhibition of multiple enzymes of the arachidonic acid cascade leads to synergistic anti-inflammatory effects. Merging of 5-lipoxygenase (5-LOX) and soluble epoxide hydrolase (sEH) pharmacophores led to the discovery of a dual 5-LOX/sEH inhibitor, which was subsequently optimized in terms of potency toward both targets and metabolic stability. The optimized lead structure displayed cellular activity in human polymorphonuclear leukocytes, oral bioavailability, and target engagement in vivo and demonstrated profound anti-inflammatory and anti-fibrotic efficiency in a kidney injury model caused by unilateral ureteral obstruction in mice. These results pave the way for investigating the therapeutic potential of dual 5-LOX/sEH inhibitors in other inflammation- and fibrosis-related disease models.

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,β-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 Experiments with 5-LO mutants, where select cysteines had been replaced by serine, indicated that the investigated melleolides suppress 5-LO product formation via two distinct modes of action: (1) by direct interference with 5-LO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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The progression of cancer is accelerated by increased proliferation, angiogenesis, and inflammation. These processes are mediated by leukotrienes. Several cancer cell lines overexpress 5-lipoxygenase, an enzyme that converts arachidonic acid into leukotrienes. An early inhibitor of the 5-lipoxygenase pathway is Rev-5901, which, however, lacks in in vivo efficacy, as it is rapidly metabolized. We investigated the introduction of carboranes as highly hydrophobic and metabolically stable pharmacophores into lipoxygenase inhibitors. Carboranes are icosahedral boron clusters that are remarkably stable and used to increase the metabolic stability of unstable pharmaceutics without changing their biological activity. By introduction of meta-carborane into Rev-5901, the first carborane-based inhibitor of the 5-lipoxygenase pathway was obtained. We report the synthesis and inhibitory and cytotoxic behavior of these compounds toward several melanoma and colon cancer cell lines and their related anticancer mechanisms., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The arachidonic acid cascade is a key player in inflammation, and numerous well-established drugs interfere with this pathway. Previous studies have suggested that simultaneous inhibition of 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH) results in synergistic anti-inflammatory effects. In this study, a novel prototype of a dual 5-LO/sEH inhibitor KM55 was rationally designed and synthesized. KM55 was evaluated in enzyme activity assays with recombinant enzymes. Furthermore, activity of KM55 in human whole blood and endothelial cells was investigated. KM55 potently inhibited both enzymes in vitro and attenuated the formation of leukotrienes in human whole blood. KM55 was also tested in a cell function-based assay. The compound significantly inhibited the LPS-induced adhesion of leukocytes to endothelial cells by blocking leukocyte activation.

Leukotrienes (LTs) are inflammatory mediators that play a pivotal role in many diseases like asthma bronchiale, atherosclerosis and in various types of cancer. The key enzyme for generation of LTs is the 5-lipoxygenase (5-LO). Here, we present a novel putative protein isoform of human 5-LO that lacks exon 4, termed 5-LOΔ4, identified in cells of lymphoid origin, namely the Burkitt lymphoma cell lines Raji and BL41 as well as primary B and T cells. Deletion of exon 4 does not shift the reading frame and therefore the mRNA is not subjected to non-mediated mRNA decay (NMD). By eliminating exon 4, the amino acids Trp144 until Ala184 are omitted in the corresponding protein. Transfection of HEK293T cells with a 5-LOΔ4 expression plasmid led to expression of the corresponding protein which suggests that the 5-LOΔ4 isoform is a stable protein in eukaryotic cells. We were also able to obtain soluble protein after expression in E. coli and purification. The isoform itself lacks canonical enzymatic activity as it misses the non-heme iron but it still retains ATP-binding affinity. Differential scanning fluorimetric analysis shows two transitions, corresponding to the two domains of 5-LO. Whilst the catalytic domain of 5-LO WT is destabilized by calcium, addition of calcium has no influence on the catalytic domain of 5-LOΔ4. Furthermore, we investigated the influence of 5-LOΔ4 on the activity of 5-LO WT and proved that it stimulates 5-LO product formation at low protein concentrations. Therefore regulation of 5-LO by its isoform 5-LOΔ4 might represent a novel mechanism of controlling the biosynthesis of lipid mediators., Competing Interests: The authors have declared that no competing interests exist.

Background: Leukotrienes are pivotal lipid mediators in various immune and inflammatory reactions. Herein, 5-LO is a validated target. 2-Aminothiazoles, as a privileged structure, implicate known 5-LO inhibitors like ST-1083 (IC50 [polymorphonuclear leukocytes (PMNL)] = 0.68 μM), yet deep structure-activity relationships (SAR) have not been established., Materials & Methods: Compounds were synthesized via Hantzsch thiazole synthesis. Inhibitory activities were evaluated using intact PMNL and purified 5-LO together with cytotoxicity measurements in U937 cells., Results: We introduced novel functionalities at 2-, 3-, 4- and 5-position of the 2-aminothiazole scaffold and conducted bioisosteric replacement to optimize the parent scaffold. SARs of the 2-aminothiazole scaffold were deduced and extended primarily for inhibition of the 5-LO enzyme., Conclusion: SAR studies provided at least two optimized leads (ST-1853, ST-1906) with high potency (IC50 [polymorphonuclear leukocytes] = 0.05 μM), specificity and noncytotoxic behavior.

Different inflammatory diseases and allergic reactions are mediated by leukotrienes, which arise from the oxygenation of arachidonic acid catalyzed by 5-lipoxygenase (5-LO). One promising approach for an effective anti-leukotriene therapy is the inhibition of this key enzyme. This study presents the synthesis and development of a potent and direct 5-LO inhibitor based on the well characterized 5-benzylidene-2-phenylthiazolone C06, whose further pharmacological investigation was precluded due to its low solubility. Through optimization of C06, evaluation of structure-activity relationships including profound assessment of the thiazolone core and consideration of the solubility, the 5-benzyl-2-phenyl-4-hydroxythiazoles represented by 46 (ST-1829, 5-(4-chlorobenzyl)-2-p-tolylthiazol-4-ol) were developed. Compound 46 showed an improved 5-LO inhibitory activity in cell-based (IC50 values 0.14 μM) and cell-free assays (IC50 values 0.03 μM) as well as a prominent enhanced solubility. Furthermore, it kept its promising inhibitory potency in the presence of blood serum, excluding excessive binding to serum proteins. These facts combined with the non-cytotoxic profile mark a major step towards an effective anti-inflammatory therapy., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Aims: The reaction of nitric oxide and nitrite-derived species with polyunsaturated fatty acids yields electrophilic fatty acid nitroalkene derivatives (NO2-FA), which display anti-inflammatory properties. Given that the 5-lipoxygenase (5-LO, ALOX5) possesses critical nucleophilic amino acids, which are potentially sensitive to electrophilic modifications, we determined the consequences of NO2-FA on 5-LO activity in vitro and on 5-LO-mediated inflammation in vivo., Results: Stimulation of human polymorphonuclear leukocytes (PMNL) with nitro-oleic (NO2-OA) or nitro-linoleic acid (NO2-LA) (but not the parent lipids) resulted in the concentration-dependent and irreversible inhibition of 5-LO activity. Similar effects were observed in cell lysates and using the recombinant human protein, indicating a direct reaction with 5-LO. NO2-FAs did not affect the activity of the platelet-type 12-LO (ALOX12) or 15-LO-1 (ALOX15) in intact cells or the recombinant protein. The NO2-FA-induced inhibition of 5-LO was attributed to the alkylation of Cys418, and the exchange of Cys418 to serine rendered 5-LO insensitive to NO2-FA. In vivo, the systemic administration of NO2-OA to mice decreased neutrophil and monocyte mobilization in response to lipopolysaccharide (LPS), attenuated the formation of the 5-LO product 5-hydroxyeicosatetraenoic acid (5-HETE), and inhibited lung injury. The administration of NO2-OA to 5-LO knockout mice had no effect on LPS-induced neutrophil or monocyte mobilization as well as on lung injury., Innovation: Prophylactic administration of NO2-OA to septic mice inhibits inflammation and promotes its resolution by interfering in 5-LO-mediated inflammatory processes., Conclusion: NO2-FAs directly and irreversibly inhibit 5-LO and attenuate downstream acute inflammatory responses.

The enzyme 5-lipoxygenase (5-LO) catalyzes the first reactions in the biosynthesis of leukotrienes, powerful lipid mediators that are involved in several physiological and pathological processes. 5-LO activity is tightly regulated by several factors, including post translational modifications (PTMs). Phosphorylations of 5-LO by the kinases extracellular signal-regulated kinase 2 (Erk2), mitogen-activated protein kinase activated protein kinase 2 (MK2) and protein kinase A (PKA) have been described to regulate 5-LO activity. Furthermore, 5-LO phosphorylation is considered a determinant of drug candidate potency. However, no evidence on a molecular level, as can be provided by MS, has as yet been presented for these PTMs. Here, we employ a workflow including different proteolytic cleavages and phosphopeptide enrichment for detection of 5-LO phosphorylation by MALDI-MS. Proof for the known phosphorylation sites of MK2 (Ser271) and PKA (Ser523) was provided by MS after in vitro phosphorylation, but not for the postulated Erk2 site (Ser663). Detection limits have been determined for all three sites. Moreover, we identified novel tyrosine kinase target sites within 5-LO using in silico and in vitro methods. Tyr42, Tyr53 and either Tyr94 or Tyr445 were phosphorylated by the Src kinases Fgr, hematopoietic cell kinase (HCK) and Yes. To analyze the phosphorylation state in the cellular context, we created stably 5-LO-transduced Mono Mac 6 cells. Here, we only detected phospho-Ser271 by MS, whereas immunoblot analysis indicated tyrosine phosphorylation, phospho-Ser271 and phospho-Ser663. Unexpectedly, phospho-Ser271 occurred independent of cell stimulation. Taken together, we describe a method for the molecular analysis of 5-LO phosphorylation, provide insights regarding the occurrence of known phosphorylation sites partly in contrast to earlier studies and present first evidence on novel phosphosites.

5-Lipoxygenase (5-LOX) catalyzes two steps in conversion of arachidonic acid to proinflammatory leukotrienes. Lipoxygenases, including human 5-LOX, consist of an N-terminal C2-like β-sandwich and a catalytic domain. We expressed the 5-LOX domains separately, these were found to interact in the yeast two-hybrid system. The 5-LOX structure suggested association between Arg(101) in the β-sandwich and Asp(166) in the catalytic domain, due to electrostatic interaction as well as hydrogen bonds. Indeed, mutagenic replacements of these residues led to loss of two-hybrid interaction. Interestingly, when Arg(101) was mutated to Asp in intact 5-LOX, enzyme activity was increased. Thus, higher initial velocity of the reaction (vinit) and increased final amount of products were monitored for 5-LOX-R101D, at several different assay conditions. In the 5-LOX crystal structure, helix α2 and adjacent loops (including Asp(166)) of the 5-LOX catalytic domain has been proposed to form a flexible lid controlling access to the active site, and lid movement would be determined by bonding of lid residues to the C2-like β-sandwich. The more efficient catalysis following disruption of the R101-D166 ionic association supports the concept of such a flexible lid in human 5-LOX., (Copyright © 2014 Elsevier Inc. All rights reserved.)

5-Lipoxygenase (5-LO) is an important enzyme of the arachidonic acid cascade and catalyses with the help of FLAP, the 5-LO-activating protein, the formation of bioactive leukotrienes (LTs). LTs are inflammatory mediators playing a pathophysiological role in different diseases such as asthma, allergic rhinitis as well as cardiovascular diseases and certain types of cancer. Up to now, only one 5-LO inhibitor is on the market, zileuton for the treatment of asthma. With the rising number of indications for anti-LT therapy, 5-LO inhibitor drug development becomes more and more important. This MiniReview gives an update on 5-LO inhibitors currently under clinical development. Furthermore, the recent advances in the search for novel 5-lipoxygenase inhibitors with a focus on computational methods are summarized. Currently, licofelone is the compound with the highest clinical development status (completed phase III trials). 5-LO inhibitor screening programmes based on computational methods could deliver several promising drug-like new molecules. These activities can be expected to be driven by the newly resolved structure of human 5-LO in the future, enabling structure-based drug design. For the prospective drugs in late-stage clinical development, the future will show their clinical safety and efficacy in the particular diseases., (© 2013 Nordic Pharmacological Society. Published by John Wiley & Sons Ltd.)

Introduction: 5-Lipoxygenase (5-LO) is a crucial enzyme of the arachidonic acid (AA) cascade and catalyzes the formation of bioactive leukotrienes (LTs) with the help of FLAP, the 5-LO-activating protein. LTs are inflammatory mediators playing a pathophysiological role in different diseases like asthma, allergic rhinitis as well as cardiovascular diseases and certain types of cancer. With the rising number of indications for anti-LT therapy, 5-LO inhibitor drug development becomes increasingly important., Areas Covered: Here, both recent findings regarding the pathophysiological role of 5-LO and the patents claimed for 5-LO inhibitors are discussed. Focusing on direct inhibitors, several patents disclosing FLAP antagonists are also subject of this review. Novel compounds include 1,5-diarylpyrazoles, indolizines and indoles and several natural product extracts., Expert Opinion: Evaluation of the patent activities revealed only quite moderate action. Nevertheless, several auspicious drug-like molecules were disclosed. It seems that in the near future, FLAP inhibitors can be expected to enter the market for the treatment of asthma. With the resolved structure of 5-LO, structure-based drug design is now applicable. Together with the identification of downstream enzyme inhibitors and dual-targeting drugs within the AA cascade, several tools are at hand to cope with 5-LOs increasing pathophysiological roles.

Background: 5-LO is an important enzyme involved in the biosynthesis of leukotrienes, which are lipid mediators of immune and inflammation responses, with important roles in respiratory disease, cardiovascular disease, immune responses and certain types of cancer. Therefore, this enzyme has been investigated as a potential target for the treatment of these pathophysiological conditions., Results: 5-LO inhibitory potential was investigated in intact polymorphonuclear leukocytes, a cell-free assay, in human whole blood and rodent cells to both elucidate structure-activity relationships and in vitro pharmacological evaluation. Chemical modifications for lead optimization via straight forward synthesis was used to combine small polar groups, which led to a suitable candidate (IC50 [polymorphonuclear leukocytes] = 1.15 µM, IC50 [S100] = 0.29 µM) with desired in vitro biopharmaceutical profiles in terms of solubility (451.9 µg/ml) and intrinsic clearance without demonstrating any cytotoxicity., Conclusion: Compound 9l is a novel, potent and selective 5-LO inhibitor with favorable preclinical drug-like properties.

Current research leads to the assumption that drugs affecting more than one target could result in a more efficient treatment of diseases and fewer safety concerns. Administration of drugs inhibiting only one branch of the arachidonic acid cascade is usually accompanied by side effects. We therefore designed and synthesized a library of hybrid molecules incorporating an imidazo[1,2-a]pyridine and an urea moiety as novel soluble epoxide hydrolase (sEH)/5-lipoxygenase (5-LO) dual inhibitors. Evaluation of the compounds was accomplished by in vitro testing using recombinant enzyme assays.

A class of 5-lipoxygenase (5-LO) inhibitors characterized by a central 5-benzylidene-2-phenyl-thiazolinone scaffold was synthesized as a new series of molecular modifications and extensions of a previously reported series. Compounds were tested in a cell-based and a cell-free assay and furthermore evaluated for their influence on cell viability. The presented substituted thiazolinone scaffold turned out to be essential for both the 5-LO inhibitory activity and the non-cytotoxic profile. With (Z)-5-(4-methoxybenzylidene)-2-(naphthalen-2-yl)-5H-thiazol-4-one (2k, ST1237), a potent, direct, non-cytotoxic 5-LO inhibitor with IC(50) of 0.08 μM and 0.12 μM (cell-free assay and intact cells), we present a promising lead optimization and development for further investigations as novel anti-inflammatory drug., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A novel class of 5-lipoxygenase (5-LO) inhibitors characterized by a central imidazo[1,2-a]pyridine scaffold, a cyclohexyl moiety and an aromatic system, is presented. This scaffold was identified in a virtual screening study and exhibits promising inhibitory potential on the 5-LO. Here, we investigate the structure-activity relationships of this compound class. With N-cyclohexyl-6-methyl-2-(4-morpholinophenyl)imidazo[1,2-a]pyridine-3-amine (14), we identified a potent 5-LO inhibitor (IC(50)=0.16μM (intact cells) and 0.1μM (cell-free)), which may possess potential as an effective lead compound intervening with inflammatory diseases and certain types of cancer., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Human 5-lipoxygenase (5-LO) can form dimers as shown here via native gel electrophoresis, gel filtration chromatography and LILBID (laser induced liquid bead ion desorption) mass spectrometry. After glutathionylation of 5-LO by diamide/glutathione treatment, dimeric 5-LO was no longer detectable and 5-LO almost exclusively exists in the monomeric form which showed full catalytic activity. Incubation of 5-LO with diamide alone led to a disulfide-bridged dimer and to oligomer formation which displays a strongly reduced catalytic activity. The bioinformatic analysis of the 5-LO surface for putative protein-protein interaction domains and molecular modeling of the dimer interface suggests a head to tail orientation of the dimer which also explains the localization of previously reported ATP binding sites. This interface domain was confirmed by the observation that 5-LO dimer formation and inhibition of activity by diamide was largely prevented when four cysteines (C159S, C300S, C416S, C418S) in this domain were mutated to serines.

A novel class of potent direct 5-lipoxygenase (5-LO) inhibitors bearing a thiazolinone-scaffold identified by virtual screening is presented. A range of substitutions and the importance of the 2-phenyl moiety were evaluated. This series is characterized by high potency in intact polymorphonuclear leukocytes and a cell-free system, exemplified by (Z)-2-(4-chlorophenyl)-5-(4-methoxybenzylidene)-5H-thiazol-4-one (18, IC(50) = 0.28 and 0.09 μM). These disubstituted thiazolinones may possess potential for intervention with inflammatory and allergic diseases and certain cancer types.

The activity of 5-LO (5-lipoxygenase), which catalyses two initial steps in the biosynthesis of pro-inflammatory LTs (leukotrienes), is strictly regulated. One recently discovered factor, CLP (coactosin-like protein), binds 5-LO and promotes LT formation. In the present paper we report that CLP also stabilizes 5-LO and prevents non-turnover inactivation of the enzyme in vitro. Mutagenesis of tryptophan residues in the 5-LO beta-sandwich showed that 5-LO-Trp102 is essential for binding to CLP, and for CLP to support 5-LO activity. In addition, the stabilizing effect also depended on binding between CLP and 5-LO. After mutations which prevent interaction (5-LO-W102A or CLP-K131A), the protective effect of CLP was absent. A calculated 5-LO-CLP docking model indicates that CLP may bind to additional residues in both domains of 5-LO, thus possibly stabilizing the 5-LO structure. To obtain further support for binding between CLP and 5-LO in a living cell, subcellular localization of CLP and 5-LO in the monocytic cell line Mono Mac 6 was determined. In these cells, 5-LO associates with a nuclear fraction only when differentiated cells are primed with phorbol ester and stimulated with ionophore. The same pattern of redistribution was found for CLP, indicating that the two proteins associate with the nucleus in a co-ordinated fashion. The results of the present study support a role for CLP as a chaperoning scaffold factor, influencing both the stability and the activity of 5-LO.