ABSTRACT
A series of derivatives of the potent dual soluble epoxide hydrolase (sEH)/5-lipoxygenase-activating protein (FLAP) inhibitor diflapolin was designed, synthesised, and characterised. These novel compounds, which contain a benzimidazole subunit were evaluated for their inhibitory activity against sEH and FLAP. Molecular modelling tools were applied to analyse structure-activity relationships (SAR) on both targets and to predict solubility and gastrointestinal (GI) absorption. The most promising dual inhibitors of these series are 5a, 6b, and 6c.
Subject(s)
Benzimidazoles , Epoxide Hydrolases , 5-Lipoxygenase-Activating Proteins/metabolism , Benzimidazoles/pharmacology , Enzyme Inhibitors/pharmacology , Lipoxygenase Inhibitors/pharmacology , Structure-Activity RelationshipABSTRACT
Massive neutrophil infiltration is an early key event in infectious inflammation, accompanied by chemotactic leukotriene (LT)B4 generation. LTB4 biosynthesis is mediated by 5-lipoxygenase (5-LOX), but which pathogenic factors cause 5-LOX activation during bacterial infections is elusive. Here, we reveal staphylococcal exotoxins as 5-LOX activators. Conditioned medium of wild-type Staphylococcus aureus but not of exotoxin-deficient strains induced 5-LOX activation in transfected HEK293 cells. Two different staphylococcal exotoxins mimicked the effects of S. aureus-conditioned medium: (1) the pore-forming toxin α-hemolysin and (2) amphipathic α-helical phenol-soluble modulin (PSM) peptides. Interestingly, in human neutrophils, 5-LOX activation was exclusively evoked by PSMs, which was prevented by the selective FPR2/ALX receptor antagonist WRW4. 5-LOX activation by PSMs was confirmed in vivo as LT formation in infected paws of mice was impaired in response to PSM-deficient S. aureus. Conclusively, exotoxins from S. aureus are potent pathogenic factors that activate 5-LOX and induce LT formation in neutrophils.
Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , Enzyme Activation/drug effects , Exotoxins/pharmacology , Leukotrienes/biosynthesis , Staphylococcus aureus/metabolism , Animals , Bacterial Toxins/pharmacology , Calcium/metabolism , Foot Diseases/metabolism , Foot Diseases/pathology , Foot Diseases/veterinary , HEK293 Cells , Hemolysin Proteins/pharmacology , Humans , Mice , Mice, Inbred C57BL , Neutrophils/drug effects , Neutrophils/metabolism , Oligopeptides/pharmacology , Receptors, Lipoxin/metabolism , Signal Transduction/drug effects , Staphylococcal Infections/metabolism , Staphylococcal Infections/pathology , Staphylococcal Infections/veterinary , Staphylococcus aureus/pathogenicityABSTRACT
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.
Subject(s)
Chromatography, High Pressure Liquid/methods , Fatty Acids, Unsaturated/analysis , Glutathione/metabolism , Oxygen/metabolism , Tandem Mass Spectrometry/methods , Blood Platelets/metabolism , Fatty Acids, Unsaturated/blood , Fatty Acids, Unsaturated/isolation & purification , Fatty Acids, Unsaturated/metabolism , Humans , Macrophages/metabolism , Phenotype , Solid Phase ExtractionABSTRACT
The epidithiodioxopiperazine gliotoxin is a virulence factor of Aspergillus fumigatus, the most important airborne fungal pathogen of humans. Gliotoxin suppresses innate immunity in invasive aspergillosis, particularly by compromising neutrophils, but the underlying molecular mechanisms remain elusive. Neutrophils are the first responders among innate immune cells recruited to sites of infection by the chemoattractant leukotriene (LT)B4 that is biosynthesized by 5-lipoxygenase and LTA4 hydrolase (LTA4H). Here, we identified gliotoxin as inhibitor of LTA4H that selectively abrogates LTB4 formation in human leukocytes and in distinct animal models. Gliotoxin failed to inhibit the formation of other eicosanoids and the aminopeptidase activity of the bifunctional LTA4H. Suppression of LTB4 formation by gliotoxin required the cellular environment and/or reducing conditions, and only the reduced form of gliotoxin inhibited LTA4H activity. Conclusively, gliotoxin suppresses the biosynthesis of the potent neutrophil chemoattractant LTB4 by direct interference with LTA4H thereby impairing neutrophil functions in invasive aspergillosis.
Subject(s)
Aspergillosis/immunology , Aspergillus fumigatus/immunology , Epoxide Hydrolases/immunology , Gliotoxin/immunology , Leukotriene B4/immunology , Animals , Aspergillosis/microbiology , Cell Line , Female , Humans , Immunity, Innate , Leukocytes/immunology , Leukocytes/microbiology , Male , Mice , Neutrophils/immunology , Neutrophils/microbiology , Rats, WistarABSTRACT
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.
Subject(s)
Leukotrienes/metabolism , Macrophages/metabolism , Metabolome , Prostaglandins/metabolism , Adult , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cells, Cultured , Cyclooxygenase Inhibitors/pharmacology , Humans , Leukotriene Antagonists/pharmacology , Lipoxygenase/metabolism , Lipoxygenase Inhibitors/pharmacology , Macrophages/drug effects , Prostaglandin Antagonists/pharmacology , Prostaglandin-Endoperoxide Synthases/metabolismABSTRACT
A series of derivatives of the potent dual soluble epoxide hydrolase (sEH)/5-lipoxygenase-activating protein (FLAP) inhibitor diflapolin was designed, synthesized, and characterized by 1H NMR, 13C NMR, and elemental analysis. These novel compounds were biologically evaluated for their inhibitory activity against sEH and FLAP. Molecular modeling tools were applied to analyze structure-activity relationships (SAR) on both targets. Results show that even small modifications on the lead compound diflapolin markedly influence the inhibitory potential, especially on FLAP, suggesting very narrow SAR.
ABSTRACT
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.
Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , Armillaria/chemistry , Cysteine/metabolism , Lipoxygenase Inhibitors/pharmacology , Sesquiterpenes/pharmacology , A549 Cells , Dose-Response Relationship, Drug , Humans , Lipoxygenase Inhibitors/chemistry , Molecular Structure , Sesquiterpenes/chemistry , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
5-Lipoxygenase-Activating Protein Inhibitors/pharmacology , Enzyme Inhibitors/pharmacology , Epoxide Hydrolases/antagonists & inhibitors , 5-Lipoxygenase-Activating Protein Inhibitors/chemistry , 5-Lipoxygenase-Activating Proteins/metabolism , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Capillary Permeability/drug effects , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Epoxide Hydrolases/metabolism , Humans , Male , Mice , Molecular Structure , Protein TransportABSTRACT
Leukotrienes (LTs) are pro-inflammatory lipid mediators derived from arachidonic acid (AA) with roles in inflammatory and allergic diseases. The biosynthesis of LTs is initiated by transfer of AA via the 5-lipoxygenase-activating protein (FLAP) to 5-lipoxygenase (5-LO). FLAP inhibition abolishes LT formation exerting anti-inflammatory effects. The soluble epoxide hydrolase (sEH) converts AA-derived anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids (di-HETEs). Its inhibition consequently also counteracts inflammation. Targeting both LT biosynthesis and the conversion of EETs with a dual inhibitor of FLAP and sEH may represent a novel, powerful anti-inflammatory strategy. We present a pharmacophore-based virtual screening campaign that led to 20 hit compounds of which 4 targeted FLAP and 4 were sEH inhibitors. Among them, the first dual inhibitor for sEH and FLAP was identified, N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N'-(3,4-dichlorophenyl)urea with IC50 values of 200 nM in a cell-based FLAP test system and 20 nM for sEH activity in a cell-free assay.
Subject(s)
5-Lipoxygenase-Activating Proteins/metabolism , Anti-Inflammatory Agents/chemistry , Enzyme Inhibitors/chemistry , Epoxide Hydrolases/antagonists & inhibitors , 5-Lipoxygenase-Activating Protein Inhibitors/chemistry , 5-Lipoxygenase-Activating Protein Inhibitors/pharmacology , Anti-Inflammatory Agents/pharmacology , Cell-Free System , Computer Simulation , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacology , Humans , Leukotrienes/biosynthesis , Models, Molecular , Molecular StructureABSTRACT
Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2-step reaction catalyzed by 5-lipoxygenase (5-LOX) requiring the formation of 5-HPETE [5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid] and its subsequent transformation to LTA4 5-LOX is thought to receive arachidonic acid from the nuclear membrane-embedded 5-LOX-activating protein (FLAP). The crystal structure of 5-LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5-LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. For 5-LOX-F177A and 5-LOX-F177/Y181A, formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild type (wt). Strikingly, coexpression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE (5-hydroxy- and 5-peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid) by these same 5-LOX mutants (≈60-70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Yet 5-LOX-Y181A generated 5-H(p)ETE at levels comparable to 5-LOX-wt but reduced LTA4 hydrolysis products. Coexpression of FLAP partially restored LTA4 hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.-Gerstmeier, J., Newcomer, M. E., Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation.