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Here, we demonstrate a structure-based small molecule virtual screening and lead optimization pipeline using a homology model of a difficult-to-drug G-protein-coupled receptor (GPCR) target. Protease-activated receptor 4 (PAR4) is activated by thrombin cleavage, revealing a tethered ligand that activates the receptor, making PAR4 a challenging target. A virtual screen of a make-on-demand chemical library yielded a one-hit compound. From the single-hit compound, we developed a novel series of PAR4 antagonists. Subsequent lead optimization via simultaneous virtual library searches and structure-based rational design efforts led to potent antagonists of thrombin-induced activation. Interestingly, this series of antagonists was active against PAR4 activation by the native protease thrombin cleavage but not the synthetic PAR4 agonist peptide AYPGKF.
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BACKGROUND & AIMS: The amino acid hypusine, synthesized from the polyamine spermidine by the enzyme deoxyhypusine synthase (DHPS), is essential for the activity of eukaryotic translation initiation factor 5A (EIF5A). The role of hypusinated EIF5A (EIF5AHyp) remains unknown in intestinal homeostasis. Our aim was to investigate EIF5AHyp in the gut epithelium in inflammation and carcinogenesis. METHODS: We used human colon tissue messenger RNA samples and publicly available transcriptomic datasets, tissue microarrays, and patient-derived colon organoids. Mice with intestinal epithelial-specific deletion of Dhps were investigated at baseline and in models of colitis and colon carcinogenesis. RESULTS: We found that patients with ulcerative colitis and Crohn's disease exhibit reduced colon levels of DHPS messenger RNA and DHPS protein and reduced levels of EIF5AHyp. Similarly, colonic organoids from colitis patients also show down-regulated DHPS expression. Mice with intestinal epithelial-specific deletion of Dhps develop spontaneous colon hyperplasia, epithelial proliferation, crypt distortion, and inflammation. Furthermore, these mice are highly susceptible to experimental colitis and show exacerbated colon tumorigenesis when treated with a carcinogen. Transcriptomic and proteomic analysis on colonic epithelial cells demonstrated that loss of hypusination induces multiple pathways related to cancer and immune response. Moreover, we found that hypusination enhances translation of numerous enzymes involved in aldehyde detoxification, including glutathione S-transferases and aldehyde dehydrogenases. Accordingly, hypusination-deficient mice exhibit increased levels of aldehyde adducts in the colon, and their treatment with a scavenger of electrophiles reduces colitis. CONCLUSIONS: Hypusination in intestinal epithelial cells has a key role in the prevention of colitis and colorectal cancer, and enhancement of this pathway via supplementation of spermidine could have a therapeutic impact.
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Colite , Espermidina , Humanos , Animais , Camundongos , Espermidina/farmacologia , Espermidina/metabolismo , Proteômica , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Carcinogênese/genética , Colite/induzido quimicamente , Colite/genética , Colite/prevenção & controle , Homeostase , InflamaçãoRESUMO
Hantaviruses are high-priority emerging pathogens carried by rodents and transmitted to humans by aerosolized excreta or, in rare cases, person-to-person contact. While infections in humans are relatively rare, mortality rates range from 1 to 40% depending on the hantavirus species. There are currently no FDA-approved vaccines or therapeutics for hantaviruses, and the only treatment for infection is supportive care for respiratory or kidney failure. Additionally, the human humoral immune response to hantavirus infection is incompletely understood, especially the location of major antigenic sites on the viral glycoproteins and conserved neutralizing epitopes. Here, we report antigenic mapping and functional characterization for four neutralizing hantavirus antibodies. The broadly neutralizing antibody SNV-53 targets an interface between Gn/Gc, neutralizes through fusion inhibition and cross-protects against the Old World hantavirus species Hantaan virus when administered pre- or post-exposure. Another broad antibody, SNV-24, also neutralizes through fusion inhibition but targets domain I of Gc and demonstrates weak neutralizing activity to authentic hantaviruses. ANDV-specific, neutralizing antibodies (ANDV-5 and ANDV-34) neutralize through attachment blocking and protect against hantavirus cardiopulmonary syndrome (HCPS) in animals but target two different antigenic faces on the head domain of Gn. Determining the antigenic sites for neutralizing antibodies will contribute to further therapeutic development for hantavirus-related diseases and inform the design of new broadly protective hantavirus vaccines.
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Doenças Transmissíveis , Vírus Hantaan , Infecções por Hantavirus , Orthohantavírus , Animais , Humanos , Anticorpos Neutralizantes , Anticorpos Antivirais , Infecções por Hantavirus/prevenção & controle , RoedoresRESUMO
BACKGROUND & AIMS: Inflammation in the gastrointestinal tract may lead to the development of cancer. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation during the inflammatory response and form covalent adducts with amine-containing macromolecules. Thus, we sought to determine the role of dicarbonyl electrophiles in inflammation-associated carcinogenesis. METHODS: The formation of isoLG adducts was analyzed in the gastric tissues of patients infected with Helicobacter pylori from gastritis to precancerous intestinal metaplasia, in human gastric organoids, and in patients with colitis and colitis-associated carcinoma (CAC). The effect on cancer development of a potent scavenger of dicarbonyl electrophiles, 5-ethyl-2-hydroxybenzylamine (EtHOBA), was determined in transgenic FVB/N insulin-gastrin (INS-GAS) mice and Mongolian gerbils as models of H pylori-induced carcinogenesis and in C57BL/6 mice treated with azoxymethane-dextran sulfate sodium as a model of CAC. The effect of EtHOBA on mutations in gastric epithelial cells of H pylori-infected INS-GAS mice was assessed by whole-exome sequencing. RESULTS: We show increased isoLG adducts in gastric epithelial cell nuclei in patients with gastritis and intestinal metaplasia and in human gastric organoids infected with H pylori. EtHOBA inhibited gastric carcinoma in infected INS-GAS mice and gerbils and attenuated isoLG adducts, DNA damage, and somatic mutation frequency. Additionally, isoLG adducts were elevated in tissues from patients with colitis, colitis-associated dysplasia, and CAC as well as in dysplastic tumors of C57BL/6 mice treated with azoxymethane-dextran sulfate sodium. In this model, EtHOBA significantly reduced adduct formation, tumorigenesis, and dysplasia severity. CONCLUSIONS: Dicarbonyl electrophiles represent a link between inflammation and somatic genomic alterations and are thus key targets for cancer chemoprevention.
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Transformação Celular Neoplásica/imunologia , Neoplasias Associadas a Colite/imunologia , Lipídeos/imunologia , Lesões Pré-Cancerosas/imunologia , Neoplasias Gástricas/imunologia , Animais , Benzilaminas/farmacologia , Benzilaminas/uso terapêutico , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias Associadas a Colite/microbiologia , Neoplasias Associadas a Colite/patologia , Neoplasias Associadas a Colite/prevenção & controle , Modelos Animais de Doenças , Células Epiteliais , Mucosa Gástrica/citologia , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/imunologia , Mucosa Gástrica/patologia , Gastrite/imunologia , Gastrite/microbiologia , Gastrite/patologia , Gerbillinae , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/imunologia , Helicobacter pylori/isolamento & purificação , Humanos , Lipídeos/antagonistas & inibidores , Metaplasia/imunologia , Metaplasia/microbiologia , Metaplasia/patologia , Camundongos , Camundongos Transgênicos , Organoides , Lesões Pré-Cancerosas/tratamento farmacológico , Lesões Pré-Cancerosas/microbiologia , Lesões Pré-Cancerosas/patologia , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Neoplasias Gástricas/prevenção & controleRESUMO
Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension, and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products, isolevuglandins, and that scavenging of mitochondrial isolevuglandins improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isolevuglandins-protein adducts in patients with essential hypertension and Ang II (angiotensin II) model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isolevuglandins was tested by the novel mitochondria-targeted isolevuglandin scavenger, mito2HOBA. Mitochondrial isolevuglandins in arterioles from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects increased deacetylation of a key mitochondrial antioxidant, SOD2 (superoxide dismutase 2). In human aortic endothelial cells stimulated with Ang II plus TNF (tumor necrosis factor)-α, mito2HOBA reduced mitochondrial superoxide and cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In Ang II-infused mice, mito2HOBA diminished mitochondrial isolevuglandins-protein adducts, raised Sirt3 (sirtuin 3) mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in Ang II-infused mice. These data support the role of mitochondrial isolevuglandins in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isolevuglandins may have therapeutic potential in treatment of vascular dysfunction and hypertension.
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Arteríolas/fisiopatologia , Pressão Sanguínea/fisiologia , Hipertensão Essencial/fisiopatologia , Lipídeos/análise , Mitocôndrias/metabolismo , Estresse Oxidativo , Angiotensina II , Animais , Antioxidantes/metabolismo , Arteríolas/efeitos dos fármacos , Arteríolas/metabolismo , Hipertensão Essencial/induzido quimicamente , Hipertensão Essencial/metabolismo , Feminino , Sequestradores de Radicais Livres/farmacologia , Humanos , Lipídeos/antagonistas & inibidores , Masculino , Camundongos Endogâmicos C57BL , Sirtuína 3/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Lipid peroxidation generates reactive dicarbonyls including isolevuglandins (IsoLGs) and malondialdehyde (MDA) that covalently modify proteins. Humans with familial hypercholesterolemia (FH) have increased lipoprotein dicarbonyl adducts and dysfunctional HDL. We investigate the impact of the dicarbonyl scavenger, 2-hydroxybenzylamine (2-HOBA) on HDL function and atherosclerosis in Ldlr-/- mice, a model of FH. Compared to hypercholesterolemic Ldlr-/- mice treated with vehicle or 4-HOBA, a nonreactive analogue, 2-HOBA decreases atherosclerosis by 60% in en face aortas, without changing plasma cholesterol. Ldlr-/- mice treated with 2-HOBA have reduced MDA-LDL and MDA-HDL levels, and their HDL display increased capacity to reduce macrophage cholesterol. Importantly, 2-HOBA reduces the MDA- and IsoLG-lysyl content in atherosclerotic aortas versus 4-HOBA. Furthermore, 2-HOBA reduces inflammation and plaque apoptotic cells and promotes efferocytosis and features of stable plaques. Dicarbonyl scavenging with 2-HOBA has multiple atheroprotective effects in a murine FH model, supporting its potential as a therapeutic approach for atherosclerotic cardiovascular disease.
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Aterosclerose/metabolismo , Benzilaminas/metabolismo , Benzilaminas/farmacologia , Benzilaminas/uso terapêutico , Hiperlipoproteinemia Tipo II/metabolismo , Receptores de LDL/genética , Animais , Aorta , Apolipoproteínas E , Aterosclerose/tratamento farmacológico , Colesterol/sangue , Colesterol/metabolismo , Feminino , Humanos , Hiperlipoproteinemia Tipo II/tratamento farmacológico , Hiperlipoproteinemia Tipo II/patologia , Inflamação/tratamento farmacológico , Peroxidação de Lipídeos , Lipoproteínas HDL/metabolismo , Lipoproteínas IDL/sangue , Lipoproteínas IDL/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragmentos de PeptídeosRESUMO
Oxidative damage is implicated in atrial fibrillation (AF), but antioxidants are ineffective therapeutically. The authors tested the hypothesis that highly reactive lipid dicarbonyl metabolites, or isolevuglandins (IsoLGs), are principal drivers of AF during hypertension. In a hypertensive murine model and stretched atriomyocytes, the dicarbonyl scavenger 2-hydroxybenzylamine (2-HOBA) prevented IsoLG adducts and preamyloid oligomers (PAOs), and AF susceptibility, whereas the ineffective analog 4-hydroxybenzylamine (4-HOBA) had minimal effect. Natriuretic peptides generated cytotoxic oligomers, a process accelerated by IsoLGs, contributing to atrial PAO formation. These findings support the concept of pre-emptively scavenging reactive downstream oxidative stress mediators as a potential therapeutic approach to prevent AF.
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Esôfago/metabolismo , Esôfago/patologia , Refluxo Gastroesofágico/metabolismo , Refluxo Gastroesofágico/patologia , Lipídeos/química , Acetilcisteína/farmacologia , Animais , Benzilaminas/farmacologia , Ácidos e Sais Biliares/metabolismo , Linhagem Celular , Óxidos N-Cíclicos/farmacologia , Humanos , Camundongos , Marcadores de Spin , Proteína Supressora de Tumor p53/metabolismoRESUMO
Identifying preventive targets for Alzheimer's disease is a central challenge of modern medicine. Non-steroidal anti-inflammatory drugs, which inhibit the cyclooxygenase enzymes COX-1 and COX-2, reduce the risk of developing Alzheimer's disease in normal ageing populations. This preventive effect coincides with an extended preclinical phase that spans years to decades before onset of cognitive decline. In the brain, COX-2 is induced in neurons in response to excitatory synaptic activity and in glial cells in response to inflammation. To identify mechanisms underlying prevention of cognitive decline by anti-inflammatory drugs, we first identified an early object memory deficit in APPSwe-PS1ΔE9 mice that preceded previously identified spatial memory deficits in this model. We modelled prevention of this memory deficit with ibuprofen, and found that ibuprofen prevented memory impairment without producing any measurable changes in amyloid-ß accumulation or glial inflammation. Instead, ibuprofen modulated hippocampal gene expression in pathways involved in neuronal plasticity and increased levels of norepinephrine and dopamine. The gene most highly downregulated by ibuprofen was neuronal tryptophan 2,3-dioxygenase (Tdo2), which encodes an enzyme that metabolizes tryptophan to kynurenine. TDO2 expression was increased by neuronal COX-2 activity, and overexpression of hippocampal TDO2 produced behavioural deficits. Moreover, pharmacological TDO2 inhibition prevented behavioural deficits in APPSwe-PS1ΔE9 mice. Taken together, these data demonstrate broad effects of cyclooxygenase inhibition on multiple neuronal pathways that counteract the neurotoxic effects of early accumulating amyloid-ß oligomers.
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Doença de Alzheimer/prevenção & controle , Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Transtornos da Memória/prevenção & controle , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase , Modelos Animais de Doenças , Regulação para Baixo , Eletroencefalografia , Ibuprofeno , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Reconhecimento Psicológico/efeitos dos fármacos , Triptofano Oxigenase/efeitos dos fármacosRESUMO
Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts, whereas histidine and arginine do not. N(ε)-Oxopropenyllysine, a lysine-lysine cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is N(ε)-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modification of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal dramatically reduces XPA's ability to bind to a DNA substrate.
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Adenina/análogos & derivados , Albumina Sérica/química , Proteína de Xeroderma Pigmentoso Grupo A/química , Adenina/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cisteína/química , Polarização de Fluorescência , Humanos , Lisina/química , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/química , Espectrometria de Massas em TandemRESUMO
A persistent and nonresolving inflammatory response to accumulating Aß peptide species is a cardinal feature in the development of Alzheimer's disease (AD). In response to accumulating Aß peptide species, microglia, the innate immune cells of the brain, generate a toxic inflammatory response that accelerates synaptic and neuronal injury. Many proinflammatory signaling pathways are linked to progression of neurodegeneration. However, endogenous anti-inflammatory pathways capable of suppressing Aß-induced inflammation represent a relatively unexplored area. Here we report that signaling through the prostaglandin-E2 (PGE2) EP4 receptor potently suppresses microglial inflammatory responses to Aß42 peptides. In cultured microglial cells, EP4 stimulation attenuated levels of Aß42-induced inflammatory factors and potentiated phagocytosis of Aß42. Microarray analysis demonstrated that EP4 stimulation broadly opposed Aß42-driven gene expression changes in microglia, with enrichment for targets of IRF1, IRF7, and NF-κB transcription factors. In vivo, conditional deletion of microglial EP4 in APPSwe-PS1ΔE9 (APP-PS1) mice conversely increased inflammatory gene expression, oxidative protein modification, and Aß deposition in brain at early stages of pathology, but not at later stages, suggesting an early anti-inflammatory function of microglial EP4 signaling in the APP-PS1 model. Finally, EP4 receptor levels decreased significantly in human cortex with progression from normal to AD states, suggesting that early loss of this beneficial signaling system in preclinical AD development may contribute to subsequent progression of pathology.
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Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/farmacologia , Inflamação/metabolismo , Microglia/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Transdução de Sinais/fisiologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Humanos , Inflamação/patologia , Éteres Metílicos/farmacologia , Microglia/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP4/agonistas , Transdução de Sinais/efeitos dos fármacosRESUMO
Inflammation and subsequent cyclooxygenase-2 (COX-2) activity has long been linked with the development of cancer, although little is known about any epigenetic effects of COX-2. A product of COX-2 activation, levuglandin (LG) quickly forms covalent bonds with nearby primary amines, such as those in lysine, which leads to LG-protein adducts. Here, we demonstrate that COX-2 activity causes LG-histone adducts in cultured cells and liver tissue, detectable through LC-MS, with the highest incidence in histone H4. Adduction is blocked by a γ-ketoaldehyde scavenger, which has no effect on COX-2 activity as measured by PGE2 production. Formation of the LG-histone adduct is associated with an increased histone solubility in NaCl, indicating destabilization of the nucleosome structure; this is also reversed with scavenger treatment. These data demonstrate that COX-2 activity can cause histone adduction and loosening of the nucleosome complex, which could lead to altered transcription and contribute to carcinogenesis.
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Ciclo-Oxigenase 2/química , DNA/química , Histonas/química , Prostaglandina D2/análogos & derivados , Prostaglandinas E/química , Cromatografia Líquida , Espectrometria de Massas , Prostaglandina D2/química , SolubilidadeRESUMO
Acetaminophen (ApAP) is an electron donor capable of reducing radicals generated by redox cycling of hemeproteins. It acts on the prostaglandin H synthases (cyclooxygenases; COXs) to reduce the protoporphyrin radical cation in the peroxidase site of the enzyme, thus preventing the intra-molecular electron transfer that generates the Tyr385 radical required for abstraction of a hydrogen from arachidonic acid to initiate prostaglandin synthesis. Unrelated to this pharmacological action, metabolism of ApAP by CYPs yields an iminoquinone electrophile that is responsible for the hepatotoxicity, which results from high doses of the drug. We synthesized novel heterocyclic phenols predicted to be electron donors. Two of these inhibited the oxygenation of arachidonic acid by PGHS-1 and myoglobin and also were shown to be more metabolically stable and exhibited less direct cytotoxicity than acetaminophen. They are leading candidates for studies to determine whether they are free of the metabolism-based hepatotoxicity produced by acetaminophen.
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MALDI-imaging mass spectrometry (IMS) has been shown to be a powerful tool to study drug distributions in organ tissue as well as whole animal bodies. Nevertheless, not all drugs are amenable to MALDI while others may be limited by poor sensitivity poor sensitivity. The use of chemical derivatization to improve detection of small molecules by mass spectrometry techniques is well documented. To our knowledge, however, this approach has not been applied to direct tissue analysis of small organic molecules. In this manuscript, we demonstrate the use of on-tissue chemical derivatization of a small organic molecule, 3-methoxysalicylamine (3-MoSA) a scavenger of γ-ketoaldehydes. Derivatization of 3-MoSA with 1,1'-thiocarbonyldiimidazole (TCDI) results in an oxothiazolidine derivative which is detected with much greater sensitivity by MALDI than 3-MoSA itself. TCDI treatment of tissue from mice dosed with 3-MoSA allowed images to be obtained showing its spatial distribution as well as its pharmacokinetic profile in different organs. These images correlated well with results obtained from HPLC-MS/MS analyses of the same tissues. These results provide proof-of-concept that on-tissue chemical derivatization can be used to improve detection of a small organic molecule by MALDI-IMS.
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Ácidos Aminossalicílicos/química , Técnicas de Preparação Histocitológica/métodos , Imagem Molecular/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Aldeídos/química , Animais , Imidazóis/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We report the development of a sensitive liquid chromatography-tandem mass spectrometric assay to quantitate 3-methoxysalicylamine (3-MoSA) in biological samples. Derivatization with 1,1'-thiocarbonyldiimidazole followed by C(18) reverse-phase chromatography allowed the detection of both analyte and internal standard (hexylsalicylamine) using electrospray ionization and selected reaction monitoring (SRM) in positive ion mode. We monitored the transitions from m/z 196.7 to 65.1 and from m/z 250.1 to 77.1 for 3-MoSA and HxSA, respectively. The method is validated with respect to linearity (r(2)=0.995), precision (<17% RSD), recovery (100% for 3-MoSA and HxSA), and stability (77% after storage up to 7 month at -80°C). The LOD and LOQ were 16.12 and 48.87 µg/l, respectively and the LLOQ of 1 pg/ml. In addition, we used this assay to analyze the pharmacokinetics of 3-MoSA in mouse plasma and tissues following both intraperitoneal and oral administration, providing new information regarding the distribution of this compound in vivo.
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Ácidos Aminossalicílicos/análise , Ácidos Aminossalicílicos/sangue , Benzilaminas/análise , Benzilaminas/farmacocinética , Encéfalo/metabolismo , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Administração Oral , Ácidos Aminossalicílicos/administração & dosagem , Ácidos Aminossalicílicos/farmacocinética , Animais , Benzilaminas/administração & dosagem , Benzilaminas/sangue , Química Encefálica , Injeções Intraperitoneais , Modelos Lineares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição TecidualRESUMO
OBJECTIVE: Aged muscle is characterized by a defect in the ability of leucine to stimulate protein synthesis. We showed previously that antioxidant supplementation improved the anabolic response to leucine of old muscle and reduced inflammation. The aim of the present study was to determine if the positive effects observed in muscle could be related to an improvement of local muscle oxidative status. METHODS: Two groups of 20-mo-old male Wistar rats were supplemented or not with rutin, vitamin E, vitamin A, zinc, and selenium during 7 wk. We measured body weight, food intake, oxidative status in muscle and other tissues, gastrocnemius muscle proteolytic activities, and liver glutathione metabolism. RESULTS: Antioxidant supplementation had no effect on muscle antioxidant capacity, superoxide dismutase activities, and myofibrillar protein carbonyl content and induced an increase in muscle cathepsin activities. In other tissues, antioxidant supplementation increased liver glutathione (reduced plus oxidized glutathione) content, reduced oxidative damage in the liver and spleen (as measured by γ-keto-aldehyde content), and reduced heart thiobarbituric acid-reactive substances. CONCLUSION: Our results showed that the positive effects of antioxidant supplementation observed previously on the anabolic response to leucine of old muscle were not directly related to an improvement of in situ muscle oxidative status. It could result from reduced systemic inflammation/oxidative stress. The dialog between muscle and other organs should be studied more thoroughly, especially during aging.
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Envelhecimento/metabolismo , Antioxidantes/administração & dosagem , Músculo Esquelético/enzimologia , Estresse Oxidativo , Animais , Catepsinas/metabolismo , Suplementos Nutricionais , Glutationa/metabolismo , Fígado/metabolismo , Masculino , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Miofibrilas/metabolismo , Oxirredução , Peptídeo Hidrolases/metabolismo , Carbonilação Proteica , Ratos , Ratos Wistar , Baço/metabolismo , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Expression of cyclooxygenase-2 (COX-2) is associated with the development of many pathologic conditions. The product of COX-2, prostaglandin H(2) (PGH(2)), can spontaneously rearrange to form reactive gamma-ketoaldehydes called levuglandins (LGs). This gamma-ketoaldehyde structure confers a high degree of reactivity on the LGs, which rapidly form covalent adducts with primary amines of protein residues. Formation of LG adducts of proteins has been demonstrated in pathologic conditions (e.g., increased levels in the hippocampus in Alzheimer's disease) and during physiologic function (platelet activation). On the basis of knowledge that lipid modification of proteins is known to cause their translocation and to alter their function, we hypothesize that modification of proteins by LG could have functional consequences. Testing this hypothesis requires an experimental approach that discriminates between the effects of protein modification by LG and the effects of cyclooxygenase-derived prostanoids acting through their G-protein coupled receptors. To achieve this goal, we have synthesized and evaluated a series of scavengers that react with LG with a potency more than 2 orders of magnitude greater than that with the epsilon-amine of lysine. A subset of these scavengers are shown to block the formation of LG adducts of proteins in cells without inhibiting the catalytic activity of the cyclooxygenases. Ten of these selective scavengers did not produce cytotoxicity. These results demonstrate that small molecules can scavenge LGs in cells without interfering with the formation of prostaglandins. They also provide a working hypothesis for the development of pharmacologic agents that could be used in experimental animals in vivo to assess the pathophysiological contribution of levuglandins in diseases associated with cyclooxygenase up-regulation.
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Aminas/química , Prostaglandinas H/química , Prostaglandinas/biossíntese , Aminas/síntese química , Plaquetas/metabolismo , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Células Hep G2 , Humanos , Prostaglandinas H/metabolismo , Piridoxamina/químicaRESUMO
Levels of reactive γ-ketoaldehydes derived from arachidonate increase in diseases associated with inflammation and oxidative injury. To assess the biological importance of these γ-ketoaldehydes, we previously identified salicylamine as an effective γ-ketoaldehyde scavenger in vitro and in cells. To determine if salicylamine could be administered in vivo, we developed an LC/MS/MS assay to measure salicylamine in plasma and tissues. In mice, half-life (t(1/2)) was 62 minutes. Drinking water supplementation (1-10 g/L) generated tissue concentrations (10-500 µM) within the range previously shown to inhibit γ-ketoaldehydes in cells. Therefore, oral administration of salicylamine can be used to assess the contribution of γ-ketoaldehydes in animal models of disease.
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Herein we report an investigation of the efficacy of pyridine and pyrimidine analogs of acetaminophen (ApAP) as peroxyl radical-trapping antioxidants and inhibitors of enzyme-catalyzed lipid peroxidation by cyclooxygenases (COX) and lipoxygenases (LOX). In inhibited autoxidations we find that ApAP, the common analgesic and antipyretic agent, is a very good antioxidant with a rate constant for reaction with peroxyl radicals (k(inh) = 5 x 10(5) M(-1) s(-1)) that is higher than many widely-used phenolic antioxidants, such as the ubiquitous butylated hydroxytoluene (BHT). This reactivity is reduced substantially upon incorporation of nitrogen into the phenolic ring, owing to an increase in the O-H bond dissociation enthalpy of pyridinols and pyrimidinols with respect to phenols. Incorporation of nitrogen into the phenolic ring of ApAP was also found to decrease its efficacy as an inhibitor of prostaglandin biosynthesis by ovine COX-1 (oCOX-1). This is explained on the basis of an increase in its oxidation potential and its reduced reactivity as a reducing co-substrate of the peroxidase protoporphyrin. In contrast, the efficacy of ApAP as an inhibitor of lipid hydroperoxide biosynthesis by soybean LOX-1 (sLOX-1) increased upon incorporation of nitrogen into the ring, suggesting a different mechanism of inhibition dependent on the acidity of the phenolic O-H which may involve chelation of the catalytic non-heme iron atom. The greater stability of the 3-pyridinols and 5-pyrimidinols to air oxidation as compared to phenols allowed us to evaluate some electron-rich pyridinols and pyrimidinols as inhibitors of oCOX-1 and sLOX-1. While the pyridinols had the best combination of activities as antioxidants and inhibitors of oCOX-1 and sLOX-1, they were found to be more toxic than ApAP in preliminary assays in human hepatocellular carcinoma (HepG2) cell culture. The pyrimidinols, however, were up to 17-fold more reactive to peroxyl radicals and up to 25-fold better inhibitors of prostaglandin biosynthesis than ApAP, with similar cytotoxicities to HepG2 cells at high levels of exposure.
Assuntos
Acetaminofen/análogos & derivados , Peroxidação de Lipídeos/efeitos dos fármacos , Lipoxigenase/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Piridinas/química , Pirimidinas/química , Antioxidantes , Linhagem Celular Tumoral , Inibidores de Ciclo-Oxigenase/química , Humanos , Cinética , Inibidores de Lipoxigenase/química , Prostaglandinas/biossíntese , Glycine max/enzimologia , Relação Estrutura-AtividadeRESUMO
Isoketals and levuglandins are highly reactive gamma-ketoaldehydes formed by oxygenation of arachidonic acid in settings of oxidative injury and cyclooxygenase activation, respectively. These compounds rapidly adduct to proteins via lysyl residues, which can alter protein structure/function. We examined whether pyridoxamine, which has been shown to scavenge alpha-ketoaldehydes formed by carbohydrate or lipid peroxidation, could also effectively protect proteins from the more reactive gamma-ketoaldehydes. Pyridoxamine prevented adduction of ovalbumin and also prevented inhibition of RNase A and glutathione reductase activity by the synthetic gamma-ketoaldehyde, 15-E2-isoketal. We identified the major products of the reaction of pyridoxamine with the 15-E2-isoketal, including a stable lactam adduct. Two lipophilic analogues of pyridoxamine, salicylamine and 5'-O-pentylpyridoxamine, also formed lactam adducts when reacted with 15-E2-isoketal. When we oxidized arachidonic acid in the presence of pyridoxamine or its analogues, pyridoxamine-isoketal adducts were found in significantly greater abundance than the pyridoxamine-N-acyl adducts formed by alpha-ketoaldehyde scavenging. Therefore, pyridoxamine and its analogues appear to preferentially scavenge gamma-ketoaldehydes. Both pyridoxamine and its lipophilic analogues inhibited the formation of lysyl-levuglandin adducts in platelets activated ex vivo with arachidonic acid. The two lipophilic pyridoxamine analogues provided significant protection against H2O2-mediated cytotoxicity in HepG2 cells. These results demonstrate the utility of pyridoxamine and lipophilic pyridoxamine analogues to assess the potential contributions of isoketals and levuglandins in oxidant injury and inflammation and suggest their potential utility as pharmaceutical agents in these conditions.