RESUMO
Candida albicans causes opportunistic infections ranging from mucosal mycoses to life-threatening systemic infections in immunocompromised patients. During C. albicans infection, leukotrienes and prostaglandins are formed from arachidonic acid by 5-lipoxygenase (5-LOX) and cyclooxygenases, respectively to amplify inflammatory conditions, but also to initiate macrophage infiltration to achieve tissue homeostasis. Since less is known about the cellular mechanisms triggering such lipid mediator biosynthesis, we investigated the eicosanoid formation in monocyte-derived M1 and M2 macrophages, neutrophils and HEK293 cells transfected with 5-LOX and 5-LOX-activating protein (FLAP) in response to C. albicans yeast or hyphae. Leukotriene biosynthesis was exclusively induced by hyphae in neutrophils and macrophages, whereas prostaglandin E2 was also formed in response to yeast cells by M1 macrophages. Eicosanoid biosynthesis was significantly higher in M1 compared to M2 macrophages. In HEK_5-LOX/FLAP cells only hyphae activated the essential 5-LOX translocation to the nuclear membrane. Using yeast-locked C. albicans mutants, we demonstrated that hyphal-associated protein expression is critical in eicosanoid formation. For neutrophils and HEK_5-LOX/FLAP cells, hyphal wall protein 1 was identified as the essential surface protein that stimulates leukotriene biosynthesis. In summary, our data suggest that hyphal-associated proteins of C. albicans are central triggers of eicosanoid biosynthesis in human phagocytes.
Assuntos
Candida albicans , Hifas , Humanos , Células HEK293 , Eicosanoides/metabolismo , Leucotrienos/metabolismoRESUMO
According to the arousal model of vigilance, the locus coeruleus-norepinephrine (LC-NE) system modulates sustained attention over long periods by regulating physiological arousal. Recent research has proposed that transcutaneous auricular vagus nerve stimulation (taVNS) modulates indirect physiological markers of LC-NE activity, although its effects on vigilance have not yet been examined. Aiming to develop a safe and noninvasive procedure to prevent vigilance failures in prolonged tasks, the present study examined whether taVNS can mitigate vigilance loss while modulating indirect markers of LC-NE activity. Following a preregistered protocol (https://osf.io/tu2xy/), 50 participants completed three repeated sessions in a randomized order, in which either active taVNS at individualized intensity set by participant, active taVNS set at 0.5 mA for all participants, or sham taVNS, was delivered while performing an attentional and vigilance task (i.e., ANTI-Vea). Changes in salivary alpha-amylase and cortisol concentrations were measured as markers of LC-NE activity. Self-reports of feelings associated with stimulation and guessing rate of active/sham conditions supported the efficacy of the single-blind procedure. Contrary to our predictions, the observed vigilance decrement was not modulated by active taVNS. Pairwise comparisons showed a mitigation by active taVNS on cortisol reduction across time. Interestingly, Spearman's correlational analyses showed some interindividual effects of taVNS on indirect markers of LC-NE, evidenced by positive associations between changes in salivary alpha-amylase and cortisol in active but not sham taVNS. We highlight the relevance of replicating and extending the present outcomes, investigating further parameters of stimulation and its effects on other indirect markers of LC-NE activity.
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While plastics like polyethylene terephthalate can already be degraded efficiently by the activity of hydrolases, other synthetic polymers like polyurethanes (PUs) and polyamides (PAs) largely resist biodegradation. In this study, we solved the first crystal structure of the metagenomic urethanase UMG-SP-1, identified highly flexible loop regions to comprise active site residues, and targeted a total of 20 potential hot spots by site-saturation mutagenesis. Engineering campaigns yielded variants with single mutations, exhibiting almost 3- and 8-fold improved activity against highly stable N-aryl urethane and amide bonds, respectively. Furthermore, we demonstrated the release of the corresponding monomers from a thermoplastic polyester-PU and a PA (nylonâ 6) by the activity of a single, metagenome-derived urethanase after short incubation times. Thereby, we expanded the hydrolysis profile of UMG-SP-1 beyond the reported low-molecular weight carbamates. Together, these findings promise advanced strategies for the bio-based degradation and recycling of plastic materials and waste, aiding efforts to establish a circular economy for synthetic polymers.
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Inflammatory responses are orchestrated by a plethora of lipid mediators, and perturbations of their biosynthesis or degradation hinder resolution and lead to uncontrolled inflammation, which contributes to diverse pathologies. Small molecules that induce a switch from pro-inflammatory to anti-inflammatory lipid mediators are considered valuable for the treatment of chronic inflammatory diseases. Commonly used non-steroidal anti-inflammatory drugs (NSAIDs) are afflicted with side effects caused by the inhibition of beneficial prostanoid formation and redirection of arachidonic acid (AA) into alternative pathways. Multi-target inhibitors like diflapolin, the first dual inhibitor of soluble epoxide hydrolase (sEH) and 5-lipoxygenase-activating protein (FLAP), promise improved efficacy and safety but are confronted by poor solubility and bioavailability. Four series of derivatives bearing isomeric thiazolopyridines as bioisosteric replacement of the benzothiazole core and two series additionally containing mono- or diaza-isosteres of the phenylene spacer were designed and synthesized to improve solubility. The combination of thiazolo[5,4-b]pyridine, a pyridinylen spacer and a 3,5-Cl2-substituted terminal phenyl ring (46a) enhances solubility and FLAP antagonism, while preserving sEH inhibition. Moreover, the thiazolo[4,5-c]pyridine derivative 41b, although being a less potent sEH/FLAP inhibitor, additionally decreases thromboxane production in activated human peripheral blood mononuclear cells. We conclude that the introduction of nitrogen, depending on the position, not only enhances solubility and FLAP antagonism (46a), but also represents a valid strategy to expand the scope of application towards inhibition of thromboxane biosynthesis.
Assuntos
Inibidores da Proteína Ativadora de 5-Lipoxigenase , Inibidores de Lipoxigenase , Humanos , Inibidores de Lipoxigenase/farmacologia , Inibidores da Proteína Ativadora de 5-Lipoxigenase/farmacologia , Solubilidade , Leucócitos Mononucleares/metabolismo , Epóxido Hidrolases/metabolismo , Inibidores Enzimáticos/farmacologia , Anti-Inflamatórios/farmacologia , Piridinas/farmacologia , Tromboxanos , LipídeosRESUMO
The 5-lipoxygenase (5-LOX) pathway gives rise to bioactive inflammatory lipid mediators, such as leukotrienes (LTs). 5-LOX carries out the oxygenation of arachidonic acid to the 5-hydroperoxy derivative and then to the leukotriene A4 epoxide which is converted to a chemotactic leukotriene B4 (LTB4) by leukotriene A4 hydrolase (LTA4H). In addition, LTA4H possesses aminopeptidase activity to cleave the N-terminal proline of a pro-inflammatory tripeptide, prolyl-glycyl-proline (PGP). Based on the structural characteristics of LTA4H, it is possible to selectively inhibit the epoxide hydrolase activity while sparing the inactivating, peptidolytic, cleavage of PGP. In the current study, chalcogen-containing compounds, 4-(4-benzylphenyl) thiazol-2-amine (ARM1) and its selenazole (TTSe) and oxazole (TTO) derivatives were characterized regarding their inhibitory and binding properties. All three compounds selectively inhibit the epoxide hydrolase activity of LTA4H at low micromolar concentrations, while sparing the aminopeptidase activity. These inhibitors also block the 5-LOX activity in leukocytes and have distinct inhibition constants with recombinant 5-LOX. Furthermore, high-resolution structures of LTA4H with inhibitors were determined and potential binding sites to 5-LOX were proposed. In conclusion, we present chalcogen-containing inhibitors which differentially target essential steps in the biosynthetic route for LTB4 and can potentially be used as modulators of inflammatory response by the 5-LOX pathway.
Assuntos
Calcogênios , Epóxido Hidrolases , Leucotrieno A4 , Epóxido Hidrolases/metabolismo , Araquidonato 5-Lipoxigenase , Aminopeptidases/metabolismoRESUMO
Leukotrienes (LT) are lipid mediators of the inflammatory response that are linked to asthma and atherosclerosis. LT biosynthesis is initiated by 5-lipoxygenase (5-LOX) with the assistance of the substrate-binding 5-LOX-activating protein at the nuclear membrane. Here, we contrast the structural and functional consequences of the binding of two natural product inhibitors of 5-LOX. The redox-type inhibitor nordihydroguaiaretic acid (NDGA) is lodged in the 5-LOX active site, now fully exposed by disordering of the helix that caps it in the apo-enzyme. In contrast, the allosteric inhibitor 3-acetyl-11-keto-beta-boswellic acid (AKBA) from frankincense wedges between the membrane-binding and catalytic domains of 5-LOX, some 30 Å from the catalytic iron. While enzyme inhibition by NDGA is robust, AKBA promotes a shift in the regiospecificity, evident in human embryonic kidney 293 cells and in primary immune cells expressing 5-LOX. Our results suggest a new approach to isoform-specific 5-LOX inhibitor development through exploitation of an allosteric site in 5-LOX.
Assuntos
Araquidonato 5-Lipoxigenase/química , Produtos Biológicos/química , Inibidores de Lipoxigenase/química , Masoprocol/química , Triterpenos/química , Sítio Alostérico , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/metabolismo , Produtos Biológicos/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ácidos Hidroxieicosatetraenoicos/química , Ácidos Hidroxieicosatetraenoicos/metabolismo , Leucotrieno B4/química , Leucotrieno B4/metabolismo , Inibidores de Lipoxigenase/metabolismo , Masoprocol/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Triterpenos/metabolismoRESUMO
Neutrophils are the most abundant leukocytes in circulation playing a key role in acute inflammation during microbial infections. Phagocytosis, one of the crucial defence mechanisms of neutrophils against pathogens, is amplified by chemotactic leukotriene (LT)B4 , which is biosynthesized via 5-lipoxygenase (5-LOX). However, extensive liberation of LTB4 can be destructive by over-intensifying the inflammatory process. While enzymatic biosynthesis of LTB4 is well characterized, less is known about molecular mechanisms that activate 5-LOX and lead to LTB4 formation during host-pathogen interactions. Here, we investigated the ability of the common opportunistic fungal pathogen Candida albicans to induce LTB4 formation in neutrophils, and elucidated pathogen-mediated drivers and cellular processes that activate this pathway. We revealed that C. albicans-induced LTB4 biosynthesis requires both the morphological transition from yeast cells to hyphae and the expression of hyphae-associated genes, as exclusively viable hyphae or yeast-locked mutant cells expressing hyphae-associated genes stimulated 5-LOX by [Ca2+ ]i mobilization and p38 MAPK activation. LTB4 biosynthesis was orchestrated by synergistic activation of dectin-1 and Toll-like receptor 2, and corresponding signaling via SYK and MYD88, respectively. Conclusively, we report hyphae-specific induction of LTB4 biosynthesis in human neutrophils. This highlights an expanding role of neutrophils during inflammatory processes in the response to C. albicans infections.
Assuntos
Candida albicans/metabolismo , Interações Hospedeiro-Patógeno , Hifas/química , Leucotrienos/biossíntese , Neutrófilos/metabolismo , Fagocitose , Humanos , Transdução de SinaisRESUMO
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.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Araquidonato 5-Lipoxigenase/genética , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , MicroRNAs/genética , Ribonuclease III/metabolismo , TranscriptomaRESUMO
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.
Assuntos
Benzimidazóis , Epóxido Hidrolases , Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Lipoxigenase/farmacologia , Relação Estrutura-AtividadeRESUMO
Sphingosine-1-phosphate (S1P) is a versatile signaling lipid involved in the regulation of numerous cellular processes. S1P regulates cellular proliferation, migration, and apoptosis as well as the function of immune cells. S1P is generated from sphingosine (Sph), which derives from the ceramide metabolism. In particular, high concentrations of S1P are present in the blood. This originates mainly from erythrocytes, endothelial cells (ECs), and platelets. While erythrocytes function as a storage pool for circulating S1P, platelets can rapidly generate S1P de novo, store it in large quantities, and release it when the platelet is activated. Platelets can thus provide S1P in a short time when needed or in the case of an injury with subsequent platelet activation and thereby regulate local cellular responses. In addition, platelet-dependently generated and released S1P may also influence long-term immune cell functions in various disease processes, such as inflammation-driven vascular diseases. In this review, the metabolism and release of platelet S1P are presented, and the autocrine versus paracrine functions of platelet-derived S1P and its relevance in various disease processes are discussed. New pharmacological approaches that target the auto- or paracrine effects of S1P may be therapeutically helpful in the future for pathological processes involving S1P.
Assuntos
Plaquetas , Esfingosina , Plaquetas/metabolismo , Comunicação Celular , Ceramidas/metabolismo , Células Endoteliais/metabolismo , Humanos , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
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.
Assuntos
Ácido 8,11,14-Eicosatrienoico , Araquidonato 12-Lipoxigenase , Plaquetas , Glutationa , Células HEK293 , Humanos , Espectrometria de MassasRESUMO
Microsomal prostaglandin E2 synthase-1 (mPGES-1), 5-lipoxygenase (5-LO) and 5- lipoxygenase-activating protein (FLAP) are key for biosynthesis of proinflammatory lipid mediators and pharmacologically relevant drug targets. In the present study, we made an attempt to explore the role of small heteroaromatic fragments on the 4,5-diarylisoxazol-3-carboxylic acid scaffold, which are selected to interact with focused regions in the active sites of mPGES-1, 5-LO and FLAP. We report that the simple structural variations on the benzyloxyaryl side-arm of the scaffold significantly influence the selectivity against mPGES-1, 5-LO and FLAP, enabling to produce multi-target inhibitors of these protein targets, exemplified by compound 18 (IC50 mPGES-1 = 0.16 µM; IC50 5-LO = 0.39 µM) with in vivo efficacy in animal model of inflammation. The computationally modeled binding structures of these new inhibitors for three targets provide clues for rational design of modified structures as multi-target inhibitors. In conclusion, the simple synthetic procedure, and the possibility of enhancing the potency of this class of inhibitors through structural modifications pave the way for further development of new multi-target inhibitors against mPGES-1, 5-LO and FLAP, with potential application as anti-inflammatory agents.
Assuntos
Androstenóis/farmacologia , Araquidonato 5-Lipoxigenase/metabolismo , Ácidos Carboxílicos/farmacologia , Inibidores Enzimáticos/farmacologia , Prostaglandina-E Sintases/antagonistas & inibidores , Adolescente , Adulto , Idoso , Androstenóis/síntese química , Androstenóis/química , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/química , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Voluntários Saudáveis , Humanos , Pessoa de Meia-Idade , Estrutura Molecular , Prostaglandina-E Sintases/metabolismo , Relação Estrutura-Atividade , Adulto JovemRESUMO
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.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Ativação Enzimática/efeitos dos fármacos , Exotoxinas/farmacologia , Leucotrienos/biossíntese , Staphylococcus aureus/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Cálcio/metabolismo , Doenças do Pé/metabolismo , Doenças do Pé/patologia , Doenças do Pé/veterinária , Células HEK293 , Proteínas Hemolisinas/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Oligopeptídeos/farmacologia , Receptores de Lipoxinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/patologia , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/patogenicidadeRESUMO
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.
Assuntos
Leucotrienos/metabolismo , Macrófagos/metabolismo , Metaboloma , Prostaglandinas/metabolismo , Adulto , Anti-Inflamatórios não Esteroides/farmacologia , Células Cultivadas , Inibidores de Ciclo-Oxigenase/farmacologia , Humanos , Antagonistas de Leucotrienos/farmacologia , Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/farmacologia , Macrófagos/efeitos dos fármacos , Antagonistas de Prostaglandina/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
Leukotrienes (LTs) are proinflammatory mediators derived from arachidonic acid (AA), which play significant roles in inflammatory diseases. The 5-lipoxygenase-activating protein (FLAP) is an integral membrane protein, which is essential for the initial step in LT biosynthesis. The aim of this study was to discover novel and chemically diverse FLAP inhibitors for treatment of inflammatory diseases requiring anti-LT therapy. Both ligand- and structure-based approaches were applied to explain the activities of known FLAP inhibitors in relation to their predicted binding modes. We gained valuable insights into the binding modes of the inhibitors by molecular modeling and generated a multistep virtual screening (VS) workflow in which 6.2 million compounds were virtually screened, and the molecular hypotheses were validated by testing VS-hit compounds biologically. The most potent hit compounds showed significant inhibition of FLAP-dependent cellular LT biosynthesis with IC50 values in the range from 0.13 to 0.87 µM. Collectively, this study provided novel bioactive chemotypes with potential for further development as effective anti-inflammatory drugs.
Assuntos
Leucotrienos , Inibidores de Lipoxigenase , Proteínas Ativadoras de 5-Lipoxigenase , Anti-Inflamatórios , Inibidores de Lipoxigenase/farmacologia , Modelos MolecularesRESUMO
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.
Assuntos
Produtos Biológicos/química , Simulação por Computador , Descoberta de Drogas , Inibidores Enzimáticos/química , Oxirredutases , Avaliação Pré-Clínica de Medicamentos , Humanos , Oxirredutases/antagonistas & inibidores , Oxirredutases/químicaRESUMO
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.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ácidos Graxos Insaturados/análise , Glutationa/metabolismo , Oxigênio/metabolismo , Espectrometria de Massas em Tandem/métodos , Plaquetas/metabolismo , Ácidos Graxos Insaturados/sangue , Ácidos Graxos Insaturados/isolamento & purificação , Ácidos Graxos Insaturados/metabolismo , Humanos , Macrófagos/metabolismo , Fenótipo , Extração em Fase SólidaRESUMO
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.
Assuntos
Bioensaio/métodos , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Sistema Livre de Células , Cromatografia Líquida de Alta Pressão , Células HEK293 , Humanos , Leucotrieno C4/metabolismo , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Ligação Proteica/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Espectrometria de Massas em TandemRESUMO
5-Lipoxygenase (5-LO) catalyzes the initial steps in the biosynthesis of proinflammatory leukotrienes. Upon cell activation, 5-LO translocates to the nuclear membrane where arachidonic acid is transferred by 5-LO-activating protein (FLAP) to 5-LO for metabolism. Although previous data indicate association of 5-LO with FLAP, the in situ assembly of native 5-LO/FLAP complexes remains elusive. Here, we show time-resolved 5-LO/FLAP colocalization by immunofluorescence microscopy and in situ 5-LO/FLAP interaction by proximity ligation assay at the nuclear membrane of Ca(2+)-ionophore A23187-activated human monocytes and neutrophils in relation to 5-LO activity. Although 5-LO translocation and product formation is completed within 1.5-3 min, 5-LO/FLAP interaction is delayed and proceeds up to 30 min. Though monocytes and neutrophils contain comparable amounts of 5-LO protein, neutrophils produce 3-5 times higher levels of 5-LO products due to prolonged activity, accompanied by delayed 5-LO nuclear membrane translocation. Arachidonic acid seemingly acts as adaptor for 5-LO/FLAP assembly, whereas FLAP inhibitors (MK886, 100 nM; BAY X 1005, 3 µM) disrupt the complex. We conclude that FLAP may regulate 5-LO activity in 2 ways: first by inducing an initial flexible association for efficient 5-LO product synthesis, followed by the formation of a tight 5-LO/FLAP complex that terminates 5-LO activity.
Assuntos
Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Leucócitos/metabolismo , Adulto , Ácido Araquidônico/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Células HEK293 , Humanos , Ligação Proteica , Transporte ProteicoRESUMO
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.