Highly potent and selective 5-lipoxygenase inhibition by new, simple heteroaryl-substituted catechols for treatment of inflammation.

5-Lipoxygenase (LO) catalyzes the first steps in the formation of pro-inflammatory leukotrienes (LT) that are pivotal lipid mediators contributing to allergic reactions and inflammatory disorders. Based on its key role in LT biosynthesis, 5-LO is an attractive drug target, demanding for effective and selective inhibitors with efficacy in vivo, which however, are still rare. Encouraged by the recent identification of the catechol 4-(3,4-dihydroxyphenyl)dibenzofuran 1 as 5-LO inhibitor, simple structural modifications were made to yield even more effective and selective catechol derivatives. Within this new series, the two most potent compounds 3,4-dihydroxy-3'-phenoxybiphenyl (6b) and 2-(3,4-dihydroxyphenyl)benzo[b]thiophene (6d) potently inhibited human 5-LO in cell-free (IC506b and 6d = 20 nM) and cell-based assays (IC506b = 70 nM, 6d = 60 nM). Inhibition of 5-LO was reversible, unaffected by exogenously added substrate arachidonic acid, and not primarily mediated via radical scavenging and antioxidant activities. Functional 5-LO mutants expressed in HEK293 cells were still prone to inhibition by 6b and 6d, and docking simulations revealed distinct binding of the catechol moiety to 5-LO at an allosteric site. Analysis of 5-LO nuclear membrane translocation and intracellular Ca2+ mobilization revealed that these 5-LO-activating events are hardly affected by the catechols. Importantly, the high inhibitory potency of 6b and 6d was confirmed in human blood and in a murine zymosan-induced peritonitis model in vivo. Our results enclose these novel catechol derivatives as highly potent, novel type inhibitors of 5-LO with high selectivity and with marked effectiveness under pathophysiological conditions.

Design and synthesis of functionalized 4-aryl-Catechol derivatives as new antiinflammtory agents with in vivo efficacy.

Oxidative stress and inflammation are two conditions that coexist in many multifactorial diseases and the discovery of antioxidants is an attractive approach that can simultaneously tackle two or more therapeutic targets of the arachidonic acid cascade. We report that the simple structural variations on the 4-aryl-benzene-1,2-diol side-arm of the scaffold significantly influence the selectivity against 5-LOX vs 12- and 15-LOX. Derivatives 4 a-l were evaluated for their antioxidant activity, using the DPPH, and ferric ion reducing antioxidant power (FRAP) methods. Docking simulations proposed concrete binding of the catechol series to 5-LO. Selected active compound 4-(3,4-dihydroxyphenyl)dibenzofuran (4l) was also tested in different in vivo mouse models of inflammation. 4l (0.1 mg/kg; i.p.) impaired (I) bronchoconstriction in ovalbumin-sensitized mice challenged with acetylcholine, (II) exudate formation in carrageenan-induced paw edema, and (III) zymosan-induced leukocyte infiltration in air pouches. These results pave the way for investigating the therapeutic potential of 4-aryl-benzene-1,2-diol, as novel multitarget therapeutic drugs, able to regulate the complex inflammatory cascade mechanisms.

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.

Sex differences in prostaglandin biosynthesis in neutrophils during acute inflammation.

The severity and course of inflammatory processes differ between women and men, but the biochemical mechanisms underlying these sex differences are elusive. Prostaglandins (PG) and leukotrienes (LT) are lipid mediators linked to inflammation. We demonstrated superior LT biosynthesis in human neutrophils and monocytes, and in mouse macrophages from females, and we confirmed these sex differences in vivo where female mice produced more LTs during zymosan-induced peritonitis versus males. Here, we report sex differences in PG production in neutrophils during acute inflammation. In the late phase (4-8 hrs) of mouse zymosan-induced peritonitis and rat carrageenan-induced pleurisy, PG levels in males were higher versus females, seemingly due to higher PG production in infiltrated neutrophils. Accordingly, human neutrophils from males produced more PGE2 than cells from females. Increased PG biosynthesis in males was accompanied by elevated cyclooxygenase (COX)-2 expression connected to increased nuclear factor-kappa B activation, and was abolished when LT synthesis was pharmacologically blocked, suggesting that elevated PG production in males might be caused by increased COX-2 expression and by shunting phenomena due to suppressed LT formation. Conclusively, our data reveal that the biosynthesis of pro-inflammatory PGs and LTs is conversely regulated by sex with consequences for the inflammatory response.

Evaluation of Dual 5-Lipoxygenase/Microsomal Prostaglandin E2 Synthase-1 Inhibitory Effect of Natural and Synthetic Acronychia-Type Isoprenylated Acetophenones.

Among the pathways responsible for the development of inflammatory responses, the cyclooxygenase and lipoxygenase pathways are among the most important ones. Two key enzymes, namely, 5-LO and mPGES-1, are involved in the biosynthesis of leukotrienes and prostaglandins, respectively, which are considered attractive therapeutic targets, so their dual inhibition might be an effective strategy to control inflammatory deregulation. Several natural products have been identified as 5-LO inhibitors, with some also being dual 5-LO/mPGES-1 inhibitors. Here, some prenylated acetophenone dimers from Acronychia pedunculata have been identified for their dual inhibitory potency toward 5-LO and mPGES-1. To gain insight into the SAR of this family of natural products, the synthesis and biological evaluation of analogues are presented. The results show the ability of the natural and synthetic molecules to potently inhibit 5-LO and mPEGS-1 in vitro. The potency of the most active compound (10) has been evaluated in vivo in an acute inflammatory mouse model and displayed potent anti-inflammatory activity comparable in potency to the drug zileuton used as a positive control.

Optimization of benzoquinone and hydroquinone derivatives as potent inhibitors of human 5-lipoxygenase.

Aiming to assess the biological activities of synthetic 1,4-benzoquinones, we previously synthesized different libraries of benzoquinones with lipophilic and bulky alkyl- or aryl-substituents that inhibited 5-lipoxygenase (5-LO). The high potency of 4,5-dimethoxy-3-alkyl-1,2-benzoquinones on 5-LO led to the idea to further modify the structures and thus to improve the inhibitory potential in vitro and in vivo as well as to investigate SARs. Systematic structural optimization through accurate structure-based design resulted in compound 30 (3-tridecyl-4,5-dimethoxybenzene-1,2-diol), an ubiquinol derivative that exhibited the strongest anti-inflammatory effect, with a 10-fold improved 5-LO inhibitory activity (IC50 = 28 nM) in activated neutrophils. Moreover, 30 significantly reduced inflammatory reactions in the carrageenan-induced mouse paw oedema and in zymosan-induced peritonitis in mice. Compound 30 (1 mg/kg, i.p.) potently suppressed the levels of cysteinyl-LTs 30 min after zymosan, outperforming zileuton at a dose of 10 mg/kg. The binding patterns of the quinone- and hydroquinone-based 5-LO inhibitors were analyzed by molecular docking. Together, we elucidated the optimal alkyl chain pattern of quinones and corresponding hydroquinones and reveal a series of highly potent 5-LO inhibitors with effectiveness in vivo that might be useful as anti-inflammatory drugs.

The 5-lipoxygenase inhibitor RF-22c potently suppresses leukotriene biosynthesis in cellulo and blocks bronchoconstriction and inflammation in vivo.

5-Lipoxygenase (5-LO) catalyzes the first two steps in leukotriene (LT) biosynthesis. Because LTs play pivotal roles in allergy and inflammation, 5-LO represents a valuable target for anti-inflammatory drugs. Here, we investigated the molecular mechanism, the pharmacological profile, and the in vivo effectiveness of the novel 1,2-benzoquinone-featured 5-LO inhibitor RF-22c. Compound RF-22c potently inhibited 5-LO product synthesis in neutrophils and monocytes (IC50⩾22nM) and in cell-free assays (IC50⩾140nM) without affecting 12/15-LOs, cyclooxygenase (COX)-1/2, or arachidonic acid release, in a specific and reversible manner, supported by molecular docking data. Antioxidant or iron-chelating properties were not evident for RF-22c and 5-LO-regulatory cofactors like Ca(2+) mobilization, ERK-1/2 activation, and 5-LO nuclear membrane translocation and interaction with 5-LO-activating protein (FLAP) were unaffected. RF-22c (0.1mg/kg; i.p.) impaired (I) bronchoconstriction in ovalbumin-sensitized mice challenged with acetylcholine, (II) exudate formation in carrageenan-induced paw edema, and (III) zymosan-induced leukocyte infiltration in air pouches. Taken together, RF-22c is a highly selective and potent 5-LO inhibitor in intact human leukocytes with pronounced effectiveness in different models of inflammation that warrants further preclinical analysis of this agent as anti-inflammatory drug.

Exploring the role of chloro and methyl substitutions in 2-phenylthiomethyl-benzoindole derivatives for 5-LOX enzyme inhibition.

Following the results we previously reported on a series of ethyl 2-phenylthiomethyl 5-hydroxyindole-3-carboxylate derivatives as 5-lipoxygenase (5-LOX) inhibitors, in order to obtain a more selective compound with respect to the previous generation of derivatives, we decided to modify the structure of the core ligand. The first level of structural modification involved the annelation of benzene to the indole, yielding corresponding benzo[g]indole derivatives, systematic optimization of methyl or chlorine groups in meta-, ortho- and ortho/para-position of 2-phenylthiomethyl moiety were applied. The reported results show that extension of the aromatic core led to a great enhancement of activity, especially in cell-free assay, and the accurate structure-based design provided compounds 6f, 6g and 6l that block 5-LOX activity in cell-free assays with IC50 ranging from 0.17 to 0.22 µM, and suppress 5-LOX product synthesis in polymorphonuclear leukocytes with IC50 ranging from 0.19 to 0.37 µM. Moreover we have identified 6f and 6l as dual 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors and compound 6l significantly reduces inflammatory reactions in the carrageenan-induced mouse paw oedema. The reported in vivo analysis, together with the accessible synthetic procedure, stimulate for the generation of further potent antinflammatory benzoindoles-based agents.

Human serum determination and in vitro anti-inflammatory activity of the vitamin E metabolite α-(13'-hydroxy)-6-hydroxychroman.

Cytochrome P450-derived long-chain metabolites are gaining increasing interest as bioactive intermediates of vitamin E. In this study we first report on the HPLC-ECD and GC-MS analysis in human serum of the earliest metabolite of this vitamin, namely α-(13'-hydroxy)-6-hydroxychroman (α-13'-OH). The two chromatographic procedure are sensitive enough (LOQ of 10nM) to measure α-13'-OH after hexane extraction of 1 ml of sample obtained from healthy volunteers supplemented for 1-week with 1000 IU/d (671 mg/d) RRR-α-tocopherol. The observed concentrations ranged between 15 and 50 nM, with minor differences between fasting and 4-hr post-meal state. Baseline (non-supplemented state) levels of 7.2 ± 1.6 nM were observed extracting higher volumes of serum. Biological effects of α-13'-OH investigated for the first time in RAW264.7 murine macrophages involved transcriptional control of inflammatory cytokines, and transcriptional and functional regulation of COX2 and iNOS enzymes in response to lipopolysaccharides. In conclusion, here we present the first quantitative evaluation of serum α-13'-OH also providing early evidence of the anti-inflammatory potential of this metabolite that is worth of further investigation in the area of functional and nutraceutical implications of vitamin E metabolism.

α-Tocopherol long-chain metabolite α-13'-COOH affects the inflammatory response of lipopolysaccharide-activated murine RAW264.7 macrophages.

SCOPE: Inflammatory response of macrophages is regulated by vitamin E forms. The long-chain metabolite α-13'-carboxychromanol (α-13'-COOH) is formed by hepatic α-tocopherol (α-TOH) catabolism and acts as a regulatory metabolite via pathways that are different from its metabolic precursor. METHODS AND RESULTS: Using semisynthetically-derived α-13'-COOH we profiled its action on LPS-induced expression of pro- and anti-inflammatory genes using RT-qPCR and of key proteins by Western blotting. Effects on inflammatory response were assessed by measuring production of nitric oxide and prostaglandin (PG) E2 , PGD2 , and PGF2α. α-13'-COOH inhibits proinflammatory pathways in LPS-stimulated RAW264.7 macrophages more efficiently than α-TOH. Profiling inflammation-related genes showed significant blocking of interleukin (Il)1ß by the metabolite and its precursor as well, while upregulation of Il6 was not impaired. However, induction of Il10, cyclooxygenase 2 (Cox2) and inducible nitric oxide synthase (iNos) by LPS and consequently the formation of nitric oxide and PG was significantly reduced by α-13'-COOH. Interestingly, α-13'-COOH acted independently from translocation of NFκB subunit p65. CONCLUSION: Our study sheds new light on the mode of action of α-TOH on the inflammatory response in macrophages, which may be mediated in vivo at least in part by its metabolite α-13'-COOH. Our data show that α-13'-COOH is a potent anti-inflammatory molecule.

Novel series of benzoquinones with high potency against 5-lipoxygenase in human polymorphonuclear leukocytes.

5-Lipoxygenase (5-LO) is a potential target for pharmacological intervention with various inflammatory and allergic diseases. Starting from the natural dual 5-LO/microsomal prostaglandin E2 synthase (mPGES)-1 inhibitor embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone, 2) that suppresses 5-LO activity in human primary leukocytes with IC50 = 0.8-2 µM, we synthesized 48 systematically modified derivatives of 2. We modified the 1,4-quinone to 1,2-quinone, mono- or bimethylated the hydroxyl groups, and varied the C11-n-alkyl residue (C4- to C16-n-alkyl or prenyl) of 2. Biological evaluation yields potent analogues being superior over 2 and obvious structure-activity relationships (SAR) for inhibition of 5-LO. Interestingly, conversion to 1,2-benzoquinone and bimethylation of the hydroxyl moieties strongly improves 5-LO inhibition in polymorphonuclear leukocytes versus 2 up to 60-fold, exemplified by the C12-n-alkyl derivative 22c (4,5-dimethoxy-3-dodecyl-1,2-benzoquinone) with IC50 = 29 nM. Regarding inhibition of mPGES-1, none of the novel benzoquinones could outperform the parental compound 2 (IC50 = 0.21 µM), and only modest suppressive effects on 12- and 15-LOs were evident. Together, our detailed SAR study reveals 22c as highly potent 5-LO-selective lead compound in intact cells that warrants further preclinical evaluation as anti-inflammatory agent.

Further studies on ethyl 5-hydroxy-indole-3-carboxylate scaffold: design, synthesis and evaluation of 2-phenylthiomethyl-indole derivatives as efficient inhibitors of human 5-lipoxygenase.

5-Lipoxygenase (5-LO), an enzyme that catalyzes the initial steps in the biosynthesis of pro-inflammatory leukotrienes, is an attractive drug target for the pharmacotherapy of inflammatory and allergic diseases. Here, we present the design, synthesis and biological evaluation of novel series of ethyl 5-hydroxyindole-3-carboxylate derivatives that efficiently inhibit human 5-LO. SAR analysis revealed that the potency of compounds is closely related to the positioning of the substituents at the phenylthiomethyl ring. The introduction of methyl or chlorine groups in ortho- and ortho/para-position of thiophenol represent the most favorable modifications. Among all tested compounds, ethyl 5-hydroxy-2-(mesitylthiomethyl)-1-methyl-1H-indole-3-carboxylate (19) is the most potent derivative which blocks 5-LO activity in cell-free assays with IC50 = 0.7 µM, and suppressed 5-LO product synthesis in polymorphonuclear leukocytes with IC50 = 0.23 µM.

Discovery and biological evaluation of novel 1,4-benzoquinone and related resorcinol derivatives that inhibit 5-lipoxygenase.

5-Lipoxygenase (5-LO), an enzyme that catalyzes the initial steps in the biosynthesis of pro-inflammatory leukotrienes, is an attractive drug target for the pharmacotherapy of inflammatory and allergic diseases. Here, we present the discovery and biological evaluation of novel series of 1,4-benzoquinones and respective resorcinol derivatives that efficiently inhibit human 5-LO, with little effects on other human lipoxygenases. SAR analysis revealed that the potency of the compounds strongly depends on structural features of the lipophilic residues, where bulky naphthyl or dibenzofuran moieties favor 5-LO inhibition. Among the 1,4-benzoquinones, compound Ig 5-[(2-naphthyl)methyl]-2-hydroxy-2,5-cyclohexadiene-1,4-dione potently blocked 5-LO activity in cell-free assays with IC50 = 0.78 µM, and suppressed 5-LO product synthesis in polymorphonuclear leukocytes with IC50 = 2.3 µM. Molecular docking studies suggest a concrete binding site for Ig in 5-LO where select π-π interactions along with hydrogen bond interactions accomplish binding to the active site of the enzyme. Together, our study reveals novel valuable 5-LO inhibitors with potential for further preclinical assessment as anti-inflammatory compounds.