RESUMO
Retrotransposons compose a staggering 40% of the mammalian genome. Among them, endogenous retroviruses (ERV) represent sequences that closely resemble the proviruses created from exogenous retroviral infection. ERVs make up 8 to 10% of human and mouse genomes and range from evolutionarily ancient sequences to recent acquisitions. Studies in Drosophila have provided a causal link between genomic retroviral elements and cognitive decline; however, in mammals, the role of ERVs in learning and memory remains unclear. Here we studied 2 independent murine models for ERV activation: muMT strain (lacking B cells and antibody production) and intracerebroventricular injection of streptozotocin (ICVI-STZ). We conducted behavioral assessments (contextual fear memory and spatial learning), as well as gene and protein analysis (RNA sequencing, PCR, immunohistochemistry, and western blot assays). Mice lacking mitochondrial antiviral-signaling protein (MAVS) and mice lacking stimulator of IFN genes protein (STING), 2 downstream sensors of ERV activation, provided confirmation of ERV impact. We found that muMT mice and ICVI-STZ mice induced hippocampal ERV activation, as shown by increased gene and protein expression of the Gag sequence of the transposable element intracisternal A-particle. ERV activation was accompanied by significant hippocampus-related memory impairment in both models. Notably, the deficiency of the MAVS pathway was protective against ICVI-STZ-induced cognitive pathology. Overall, our results demonstrate that ERV activation is associated with cognitive impairment in mice. Moreover, they provide a molecular target for strategies aimed at attenuating retroviral element sensing, via MAVS, to treat dementia and neuropsychiatric disorders.
Assuntos
Retrovirus Endógenos/genética , Hipocampo/virologia , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Transtornos da Memória/virologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Comportamento Animal , Encéfalo/patologia , Disfunção Cognitiva , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Retrovirus Endógenos/fisiologia , Regulação da Expressão Gênica , Produtos do Gene gag , Hipocampo/efeitos dos fármacos , Aprendizagem , Masculino , Proteínas de Membrana/metabolismo , Memória , Transtornos da Memória/psicologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estreptozocina/farmacologiaRESUMO
BACKGROUND: Extracellular high mobility group box 1 protein (HMGB1) serves a central role in inflammation as a transporter protein, which binds other immune-activating molecules that are endocytosed via the receptor for advanced glycation end-products (RAGE). These pro-inflammatory complexes are targeted to the endolysosomal compartment, where HMGB1 permeabilizes the lysosomes. This enables HMGB1-partner molecules to avoid degradation, to leak into the cytosol, and to reach cognate immune-activating sensors. Lipopolysaccharide (LPS) requires this pathway to generate pyroptosis by accessing its key cytosolic receptors, murine caspase 11, or the human caspases 4 and 5. This lytic, pro-inflammatory cell death plays a fundamental pathogenic role in gram-negative sepsis. The aim of the study was to identify molecules inhibiting HMGB1 or HMGB1/LPS cellular internalization. METHODS: Endocytosis was studied in cultured macrophages using Alexa Fluor-labeled HMGB1 or complexes of HMGB1 and Alexa Fluor-labeled LPS in the presence of an anti-HMGB1 monoclonal antibody (mAb), recombinant HMGB1 box A protein, acetylcholine, the nicotinic acetylcholine receptor subtype alpha 7 (α7 nAChR) agonist GTS-21, or a dynamin-specific inhibitor of endocytosis. Images were obtained by fluorescence microscopy and quantified by the ImageJ processing program (NIH). Data were analyzed using student's t test or one-way ANOVA followed by the least significant difference or Tukey's tests. RESULTS: Anti-HMGB1 mAb, recombinant HMGB1 antagonist box A protein, acetylcholine, GTS-21, and the dynamin-specific inhibitor of endocytosis inhibited internalization of HMGB1 or HMGB1-LPS complexes in cultured macrophages. These agents prevented macrophage activation in response to HMGB1 and/or HMGB1-LPS complexes. CONCLUSION: These results demonstrate that therapies based on HMGB1 antagonists and the cholinergic anti-inflammatory pathway share a previously unrecognized molecular mechanism of substantial clinical relevance.
Assuntos
Proteína HMGB1/metabolismo , Lipopolissacarídeos/farmacologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Acetilcolina/farmacologia , Animais , Células Cultivadas , Agonistas Colinérgicos/farmacologia , Endocitose/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Células RAW 264.7RESUMO
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in a broad range of inflammatory and oncologic diseases. MIF is unique among cytokines in terms of its release profile and inflammatory role, notably as an endogenous counter-regulator of the anti-inflammatory effects of glucocorticoids. In addition, it exhibits a catalytic tautomerase activity amenable to the design of high affinity small molecule inhibitors. Although several classes of these compounds have been identified, biologic characterization of these molecules remains a topic of active investigation. In this study, we used in vitro LPS-driven assays to characterize representative molecules from several classes of MIF inhibitors. We determined that MIF inhibitors exhibit distinct profiles of anti-inflammatory activity, especially with regard to TNFα. We further investigated a molecule with relatively low anti-inflammatory activity, compound T-614 (also known as the anti-rheumatic drug iguratimod), and found that, in addition to exhibiting selective MIF inhibition in vitro and in vivo, iguratimod also has additive effects with glucocorticoids. Furthermore, we found that iguratimod synergizes with glucocorticoids in attenuating experimental autoimmune encephalitis, a model of multiple sclerosis. Our work identifies iguratimod as a valuable new candidate for drug repurposing to MIF-relevant diseases, including multiple sclerosis.
Assuntos
Cromonas/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Glucocorticoides/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Esclerose Múltipla/tratamento farmacológico , Sulfonamidas/farmacologia , Animais , Linhagem Celular Tumoral , Cromonas/agonistas , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/metabolismo , Glucocorticoides/agonistas , Humanos , Oxirredutases Intramoleculares/metabolismo , Lipopolissacarídeos/toxicidade , Fatores Inibidores da Migração de Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Sulfonamidas/agonistas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A new synthetic protocol provides a simple and direct method to generate functionalized ß-hydroxy-tetrahydroquinolines (THQs). Hydroboration of quinolines using chloroboranes followed by oxidation with NaBO3 â H2 O led to the formation of functionalized ß-hydroxy THQs. High regio- and diastereoselectivities were observed in α and γ substituted quinolines and the trans diastereomer of the ß-hydroxy-THQ was the major isostere. This new protocol was utilized to build the novel antibody-targeted lupus peptidomimetic, FISLE-412.
Assuntos
Boranos/química , Quinolinas/química , Oxirredução , Peptidomiméticos , Quinolinas/síntese química , EstereoisomerismoRESUMO
FISLE-412 is the first reported small molecule peptidomimetic that neutralizes anti-dsDNA autoantibodies associated with systemic lupus erythematosus (SLE) pathogenesis. FISLE-412 is a complex small molecule that involves a challenging synthesis scheme, but has attractive pharmacological activities as a potential small molecule therapeutic in lupus. Therefore, we initiated a Structure-Activity Relationship study to simplify the complexity of FISLE-412. We synthesized a small library of mimetopes around the FISLE-412 structure and identified several analogues which could neutralize anti-DNA lupus antibodies in vitro and ex vivo. Our strategies reduced the structural complexity of FISLE-412 and provide important information that may guide development of potential autoantibody-targeted lupus therapeutics.
Assuntos
Anticorpos Antinucleares/metabolismo , Quinolinas/química , Saquinavir/química , Anticorpos Antinucleares/química , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Rim/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Peptidomiméticos , Poliaminas/química , Poliaminas/metabolismo , Quinolinas/metabolismo , Relação Estrutura-AtividadeRESUMO
Immune suppression by regulatory T cells and regulatory B cells is a critical mechanism to limit excess inflammation and autoimmunity. IL-10 is considered the major mediator of B cell-induced immune suppression. We report a novel mechanism for immune suppression through adenosine generation by B cells. We identified a novel population of B cells that expresses CD73 as well as CD39, two ectoenzymes that together catalyze the extracellular dephosphorylation of adenine nucleotides to adenosine. Whereas CD39 expression is common among B cells, CD73 expression is not. Approximately 30-50% of B-1 cells (B220(+)CD23(-)) and IL-10-producing B (B10) cells (B220(+)CD5(+)CD1d(hi)) are CD73(hi), depending on mouse strain, whereas few conventional B-2 cells (B220(+)CD23(+)AA4.1(-)) express CD73. In keeping with expression of both CD73 and CD39, we found that CD73(+) B cells produce adenosine in the presence of substrate, whereas B-2 cells do not. CD73(-/-) mice were more susceptible to dextran sulfate sodium salt (DSS)-induced colitis than wild type (WT) mice were, and transfer of CD73(+) B cells ameliorated the severity of colitis, suggesting that B cell CD73/CD39/adenosine can modulate DSS-induced colitis. IL-10 production by B cells is not affected by CD73 deficiency. Interestingly, adenosine generation by IL-10(-/-) B cells is impaired because of reduced expression of CD73, indicating an unexpected connection between IL-10 and adenosine and suggesting caution in interpreting the results of studies with IL-10(-/-) cells. Our findings demonstrate a novel regulatory role of B cells on colitis through adenosine generation in an IL-10-independent manner.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina/biossíntese , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Imunomodulação , 5'-Nucleotidase/genética , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Apirase/genética , Apirase/metabolismo , Colite/induzido quimicamente , Colite/genética , Colite/imunologia , Colite/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Imunomodulação/genética , Imunofenotipagem , Interleucina-10/deficiência , Interleucina-10/genética , Masculino , Camundongos , Camundongos Knockout , Modelos Biológicos , Cavidade Peritoneal/citologia , FenótipoRESUMO
Inflammatory conditions characterized by excessive immune cell activation and cytokine release, are associated with bidirectional immune system-brain communication, underlying sickness behavior and other physiological responses. The vagus nerve has an important role in this communication by conveying sensory information to the brain, and brain-derived immunoregulatory signals that suppress peripheral cytokine levels and inflammation. Brain muscarinic acetylcholine receptor (mAChR)-mediated cholinergic signaling has been implicated in this regulation. However, the possibility of controlling inflammation by peripheral administration of centrally-acting mAChR agonists is unexplored. To provide insight we used the centrally-acting M1 mAChR agonist xanomeline, previously developed in the context of Alzheimer's disease and schizophrenia. Intraperitoneal administration of xanomeline significantly suppressed serum and splenic TNF levels, alleviated sickness behavior, and increased survival during lethal murine endotoxemia. The anti-inflammatory effects of xanomeline were brain mAChR-mediated and required intact vagus nerve and splenic nerve signaling. The anti-inflammatory efficacy of xanomeline was retained for at least 20h, associated with alterations in splenic lymphocyte, and dendritic cell proportions, and decreased splenocyte responsiveness to endotoxin. These results highlight an important role of the M1 mAChR in a neural circuitry to spleen in which brain cholinergic activation lowers peripheral pro-inflammatory cytokines to levels favoring survival. The therapeutic efficacy of xanomeline was also manifested by significantly improved survival in preclinical settings of severe sepsis. These findings are of interest for strategizing novel therapeutic approaches in inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Agonistas Muscarínicos/farmacologia , Piridinas/farmacologia , Sepse/prevenção & controle , Baço/fisiologia , Tiadiazóis/farmacologia , Nervo Vago/fisiologia , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anti-Inflamatórios não Esteroides/uso terapêutico , Citocinas/fisiologia , Comportamento de Doença/efeitos dos fármacos , Injeções Intraperitoneais , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos BALB C , Agonistas Muscarínicos/administração & dosagem , Agonistas Muscarínicos/uso terapêutico , Piridinas/administração & dosagem , Piridinas/uso terapêutico , Ratos Sprague-Dawley , Receptor Muscarínico M1/agonistas , Sepse/mortalidade , Baço/efeitos dos fármacos , Baço/inervação , Análise de Sobrevida , Tiadiazóis/administração & dosagem , Tiadiazóis/uso terapêutico , Fator de Necrose Tumoral alfa/sangue , VagotomiaRESUMO
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of pathogenic autoantibodies, many of which are directed against nuclear antigens, in particular double-stranded (ds) DNA. Both clinical studies and animal models have shown that anti-dsDNA antibodies contribute to kidney disease, which is present in 50% of lupus patients and is a major cause of mortality. We previously demonstrated that a subset of nephrotoxic anti-dsDNA antibodies also recognizes the pentapeptide consensus sequence D/E W D/E Y S/G (DWEYS) present in the NR2A and NR2B subunits of the N-methyl-d-aspartate receptor (NMDAR). Autoantibodies with this specificity are present in ≈40% of lupus patient sera and are both nephrotoxic and neurotoxic. Elevated titers are present in cerebrospinal fluid of patients with central nervous system manifestations of SLE. Administration of the nonnaturally occurring D form of the DWEYS pentapeptide prevents these antibodies from depositing in glomeruli and from mediating neuronal excitotoxicity. To craft a more useful therapeutic, we used the structural features of the DWEYS peptide to design a unique, selective, and potent small molecule peptidomimetic, FISLE-412, which neutralizes anti-dsDNA/NMDAR lupus autoantibodies and prevents their pathogenic interaction with tissue antigens. This compound, or others derived from it, may provide a unique strategy for the development of lupus therapeutics.
Assuntos
Autoanticorpos/imunologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Peptidomiméticos/imunologia , Peptidomiméticos/uso terapêutico , Animais , Autoanticorpos/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Peptídeos/genética , Peptídeos/imunologia , Peptidomiméticos/síntese química , Peptidomiméticos/químicaRESUMO
Abnormally low plasma concentrations of thyroid hormones during sepsis often occur in the absence of thyroidal illness; however, the mechanisms involved in the "euthyroid sick syndrome" remain poorly understood. Here, we describe a previously unrecognized interaction between the thyroid hormone thyroxine (T(4)) and the proinflammatory cytokine macrophage migration inhibitory factor (MIF), together with its clinical relevance in sepsis. We found that in both patients with severe sepsis, and our rodent model, low plasma T(4) concentrations were inversely correlated with plasma MIF concentrations. The MIF molecule contains a hydrophobic pocket that is important for many of its proinflammatory activities. Binding of L-T(4) (or its hormonally inert isomer D-T(4)) significantly, and dose-dependently, inhibited the catalytic activity of this pocket. Moreover, administration of exogenous D-T(4) significantly improved survival in mice with severe sepsis. To examine the specificity of the MIFT(4) interaction, wild-type and MIF knockout mice were subjected to the carrageenan-air pouch model of inflammation and then treated with D-T(4) or vehicle. D-T(4) significantly inhibited leukocyte infiltration in wild-type mice but not in MIF knockout mice, providing evidence that in vivo T(4) may influence MIF-mediated inflammatory responses via inhibition of its hydrophobic proinflammatory pocket. These findings demonstrate a new physiological role for T(4) as a natural inhibitor of MIF proinflammatory activity. The data may also, in part, explain the low plasma T(4) concentrations in critically ill, euthyroid patients and suggest that targeting the imbalance between MIF and T(4) may be beneficial in improving outcome from sepsis.
Assuntos
Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Tiroxina/fisiologia , Animais , Sítios de Ligação , Humanos , Inflamação , Fatores Inibidores da Migração de Macrófagos/sangue , Camundongos , Camundongos Knockout , Sepse/sangue , Sepse/tratamento farmacológico , Taxa de Sobrevida , Hormônios Tireóideos/sangue , Tiroxina/sangue , Tiroxina/uso terapêuticoRESUMO
Macrophage migration inhibitory factor (MIF), a cytokine involved in the pathogenesis of sepsis, diabetes, asthma, arthritis, and cancer, has an enzymatic active site that has proven to be an effective target for small molecule-based inhibitors. Herein, we describe the synthesis of a novel arylazoarylmethane compound (3) from a known arylhydrazone MIF inhibitor (2). This new compound has improved stability and in vivo biological activity.
Assuntos
Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Hidrazonas/síntese química , Hidrazonas/farmacologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Animais , Carragenina , Modelos Animais de Doenças , Desenho de Fármacos , Estabilidade de Medicamentos , Inflamação/induzido quimicamente , Inflamação/enzimologia , Inflamação/prevenção & controle , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Camundongos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Pulmonary hypertension (PH) is a devastating disease leading to progressive hypoxemia, right ventricular failure, and death. Hypoxia can play a pivotal role in PH etiology, inducing pulmonary vessel constriction and remodeling. These events lead to increased pulmonary vessel wall thickness, elevated vascular resistance and right ventricular hypertrophy. The current study examined the association of the inflammatory cytokine macrophage migration inhibitory factor (MIF) with chronic lung disease and its role in the development of hypoxia-induced PH. We found that plasma MIF in patients with primary PH or PH secondary to interstitial lung disease (ILD) was significantly higher than in the control group (P = 0.004 and 0.007, respectively). MIF involvement with hypoxia-induced fibroblast proliferation was examined in both a human cell-line and primary mouse cells from wild-type (mifâº/âº) and MIF-knockout (mifâ»/â») mice. In vitro, hypoxia-increased MIF mRNA, extracellular MIF protein accumulation and cell proliferation. Inhibition of MIF inflammatory activity reduced hypoxia-induced cell proliferation. However, hypoxia only increased proliferation of mifâ»/â» cells when they were supplemented with media from mifâº/⺠cells. This growth increase was suppressed by MIF inhibition. In vivo, chronic exposure of mice to a normobaric atmosphere of 10% oxygen increased lung tissue expression of mRNA encoding MIF and accumulation of MIF in plasma. Inhibition of the MIF inflammatory active site, during hypoxic exposure, significantly reduced pulmonary vascular remodeling, cardiac hypertrophy and right ventricular systolic pressure. The data suggest that MIF plays a critical role in hypoxia-induced PH, and its inhibition may be beneficial in preventing the development and progression of the disease.
Assuntos
Hipertensão Pulmonar/sangue , Hipóxia/fisiopatologia , Fatores Inibidores da Migração de Macrófagos/sangue , Fatores Inibidores da Migração de Macrófagos/metabolismo , Adulto , Idoso , Animais , Hipóxia Celular/fisiologia , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Hipóxia/sangue , Masculino , Camundongos , Pessoa de Meia-Idade , OximetriaRESUMO
Peptides, polymers of amino acids, comprise a vital and expanding therapeutic approach. Their rapid degradation by proteases, however, represents a major limitation to their therapeutic utility and chemical modifications to native peptides have been employed to mitigate this weakness. Herein, we describe functionalized thiocarbazate scaffolds as precursors of aza-amino acids, that, upon activation, can be integrated in a peptide sequence to generate azapeptides using conventional peptide synthetic methods. This methodology facilitates peptide editing-replacing targeted amino acid(s) with aza-amino acid(s) within a peptide-to form azapeptides with preferred therapeutic characteristics (extending half-life/bioavailability, while at the same time typically preserving structural features and biological activities). We demonstrate the convenience of this azapeptide synthesis platform in two well-studied peptides with short half-lives: FSSE/P5779, a tetrapeptide inhibitor of HMGB1/MD-2/TLR4 complex formation, and bradykinin, a nine-residue vasoactive peptide. This bench-stable thiocarbazate platform offers a robust and universal approach to optimize peptide-based therapeutics.
Assuntos
Aminoácidos , Bradicinina , Meia-Vida , Peptídeo Hidrolases , EndopeptidasesRESUMO
We have recently shown that covalent attachment of the NO moiety to the HIV protease inhibitor Saquinavir (Saq) produced a qualitatively new chemical entity, named Saquinavir-NO (Saq-NO), with enhanced anticancer properties and reduced toxicity. In this study we evaluated the impact of Saq-NO on the growth of A375 human melanoma cells, as a prototype of NO-dependent cancer model. The novel compound strongly affected the in vitro and in vivo progression of A375 melanoma cell growth. The mechanism of antimelanoma action comprised dual drug activity-induction of apoptotic cell death and acquisition of melanoma cell responsiveness to TRAIL. Saq-NO-triggered apoptosis was dependent on transient AKT up-regulation and reduced pERK and iNOS expression that were observed within the first 12 h of exposure to the drug. Thereafter, however, Saq-NO up-regulated both iNOS transcription and NO endogenous synthesis and sensitized A375 cells to TRAIL. Furthermore, reduced YY1 expression was observed after 24 h of Saq-NO exposure, which correlated with increased expression of DR5. The biological relevance of this complex and powerful action of Saq-NO was consistent with the marked drug-induced inhibition of the growth of A375 xenotransplants in nude mice.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Melanoma/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/metabolismo , Saquinavir/análogos & derivados , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Masculino , Melanoma/enzimologia , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos Nus , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Saquinavir/farmacologia , Fatores de Tempo , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto , Fator de Transcrição YY1/metabolismoRESUMO
Airway remodeling is the process of airway structural change that occurs in patients with asthma in response to persistent inflammation and leads to increasing disease severity. Drugs that decrease this persistent inflammation play a crucial role in managing asthma episodes. Mice sensitized (by intraperitoneal administration) and then challenged (by inhalation) with ovalbumin (OVA) develop an extensive eosinophilic inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening, and airway wall area increase, similar to pathologies observed in human asthma. We used OVA-sensitized/challenged mice as a murine model of chronic allergic airway inflammation with subepithelial fibrosis (i.e., asthma). In this OVA mouse model, mRNA and protein of macrophage migration inhibitory factor (MIF) are upregulated, a response similar to what has been observed in the pathogenesis of acute inflammation in human asthma. We hypothesized that MIF induces transforming growth factor-ß1 (TGF-ß1) synthesis, which has been shown to play an important role in asthma and airway remodeling. To explore the role of MIF in the development of airway remodeling, we evaluated the effects of an MIF small-molecule antagonist, (S,R)3-(4-hy-droxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), on pathologies associated with the airway-remodeling process in the OVA mouse model. We found that administration of ISO-1 significantly mitigated all symptoms caused by OVA treatment. In addition, the treatment of OVA-sensitized mice with the MIF antagonist ISO-1 significantly reduced TGF-ß1 mRNA levels in pulmonary tissue and its protein level in bronchial alveolar lavage fluid supernatants. We believe the repression of MIF in the ISO-1 treatment group led to the significant suppression observed in the inflammatory responses associated with the allergen-induced lung inflammation and fibrosis in our murine asthma (OVA) model. Our results implicate a possible function of MIF in the pathogenesis of chronic asthma and suggest that MIF might be an important therapeutic target for airway remodeling.
Assuntos
Remodelação das Vias Aéreas/efeitos dos fármacos , Asma/tratamento farmacológico , Asma/fisiopatologia , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Animais , Asma/complicações , Asma/genética , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Doença Crônica , Dexametasona/farmacologia , Modelos Animais de Doenças , Feminino , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Humanos , Hipertrofia , Imuno-Histoquímica , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Pulmão/fisiopatologia , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso/efeitos dos fármacos , Músculo Liso/patologia , Ovalbumina , Pneumonia/complicações , Pneumonia/tratamento farmacológico , Pneumonia/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
In this study we evaluated the effects of the new NO donating compound (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid-nitric oxide (GIT-27NO) on the A375 human melanoma cell line. Treatment with the drug led to concentration-dependent reduction of mitochondrial respiration and number of viable cells in cultures. Decreased cell viability correlated with release and internalization of NO and was neutralized by the extracellular scavenger hemoglobin. GIT-27NO neither influenced cell division nor induced accidental or autophagic cell death. Early signs of apoptosis were observed upon coculture with the drug, and resulting in marked accumulation of hypodiploid cells, suggesting that the induction of apoptosis is one primary mode of action of the compound in A375 cells. GIT-27NO significantly inhibited the expression of the transcription repressor and apoptotic resistant factor YY1 and, in parallel, augmented the presence of total p53. The capacity of GIT-27NO to induce p53-mediated apoptosis along with inhibition of YY1 repressor in A375 melanoma cells indicates that GIT-27NO possesses an important anti-cancer pharmacological profile. The findings suggest the potential therapeutic use of GIT-27NO in the clinical setting.
Assuntos
Apoptose/efeitos dos fármacos , Melanoma/tratamento farmacológico , Doadores de Óxido Nítrico/farmacologia , Proteína Supressora de Tumor p53/efeitos dos fármacos , Acetatos , Antineoplásicos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Melanoma/patologia , Doadores de Óxido Nítrico/uso terapêutico , Oxazóis , Fator de Transcrição YY1/antagonistas & inibidoresRESUMO
A series of phenolic hydrazones were synthesized and evaluated for their inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity. Compound 7 emerged as a potent inhibitor of MIF with an IC50 of 130 nM. Compound 7 dose-dependently suppressed TNFalpha secretion from lipopolysaccharide stimulated macrophages. The therapeutic importance of the MIF inhibition by 7 is demonstrated by the significant protection from the lethality of sepsis when administration of the compound was initiated in a clinically relevant time frame.
Assuntos
Hidrazonas/síntese química , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fenóis/síntese química , Sepse/tratamento farmacológico , Animais , Linhagem Celular , Hidrazonas/química , Hidrazonas/farmacologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fenóis/química , Fenóis/farmacologia , Sepse/metabolismo , Sepse/mortalidade , Relação Estrutura-Atividade , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
Systemic lupus erythematosus is an autoimmune disease that can affect numerous tissues and is characterized by the production of nuclear antigen-directed autoantibodies (e.g., anti-dsDNA). Using a combination of virtual and ELISA-based screens, we made the intriguing discovery that several HIV-protease inhibitors can function as decoy antigens to specifically inhibit the binding of anti-dsDNA antibodies to target antigens such as dsDNA and pentapeptide DWEYS. Computational modeling revealed that HIV-protease inhibitors comprised structural features present in DWEYS and predicted that analogues containing more flexible backbones would possess preferred binding characteristics. To address this, we reduced the internal amide backbone to improve flexibility, producing new small-molecule decoy antigens, which neutralize anti-dsDNA antibodies in vitro, in situ, and in vivo. Pharmacokinetic and SLE model studies demonstrated that peptidomimetic FISLE-412,1 a reduced HIV protease inhibitor analogue, was well-tolerated, altered serum reactivity to DWEYS, reduced glomeruli IgG deposition, preserved kidney histology, and delayed SLE onset in NZB/W F1 mice.
Assuntos
Anticorpos Antinucleares/imunologia , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Animais , DNA/imunologia , Descoberta de Drogas , Feminino , Inibidores da Protease de HIV/farmacocinética , Inibidores da Protease de HIV/farmacologia , Humanos , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/imunologia , Glomérulos Renais/patologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Camundongos Endogâmicos NZB , Modelos MolecularesRESUMO
The design, self-assembly, and characterization of discrete arrays of porphyrazine (Pz) squares are presented to illustrate the supramolecular chemistry of these macrocycles, and to highlight that these arrays have physical chemical properties that are different from similar arrays of porphyrinic systems.
Assuntos
Metaloporfirinas/síntese química , Catálise , Fenômenos Químicos , Físico-Química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metaloporfirinas/química , Compostos Organoplatínicos/química , Paládio/química , Pirróis/síntese química , Espectrofotometria UltravioletaRESUMO
Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine, which possesses a contributing role in cancer progression and metastasis and, thus, is now considered a promising anticancer drug target. Many MIF-inactivating strategies have proven successful in delaying cancer growth. Here, we report on the synthesis of ISO-66, a novel, highly stable, small-molecule MIF inhibitor, an analog of ISO-1 with improved characteristics. The MIF:ISO-66 co-crystal structure demonstrated that ISO-66 ligates the tautomerase active site of MIF, which has previously been shown to play an important role in its biological functions. In vitro, ISO-66 enhanced specific and non-specific anticancer immune responses, whereas prolonged administration of ISO-66 in mice with established syngeneic melanoma or colon cancer was non-toxic and resulted in a significant decrease in tumor burden. Subsequent ex vivo analysis of mouse splenocytes revealed that the observed decrease in tumor growth rates was likely mediated by the selective in vivo expansion of antitumor-reactive effector cells induced by ISO-66. Compared to other MIF-inactivating strategies employed in vivo, the anticancer activity of ISO-66 is demonstrated to be of equal or better efficacy. Our findings suggest that targeting MIF, via highly specific and stable compounds, such as ISO-66, may be effective for cancer treatment and stimulation of anticancer immune responses.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Isoxazóis/uso terapêutico , Melanoma/tratamento farmacológico , Fenóis/uso terapêutico , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Domínio Catalítico/efeitos dos fármacos , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isoxazóis/síntese química , Isoxazóis/química , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/química , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos Nus , Neoplasias Experimentais , Fenóis/síntese química , Fenóis/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Baço/efeitos dos fármacos , Baço/metabolismoRESUMO
Macrophage migration inhibitory factor is a multifunctional cytokine involved in the regulation of immune processes and also in apoptosis induction. Elevated MIF expression is detrimental for insulin-producing beta cells and MIF inhibition protected beta cells from several cytotoxic insults such as inflammatory cytokines, high fatty acids or high glucose concentrations. Therefore, the aim of this study was to investigate two newly synthesized small molecule MIF inhibitors (K664-1 and K647-1) and to compare them with previously established effects of the prototypical MIF inhibitor, ISO-1. Our results indicate that K664-1 and K647-1 are 160- and 40-fold more effective in inhibition of MIF׳s tautomerase activity than ISO-1. Also, new inhibitors confer beta cell protection from cytokine-triggered apoptosis at significantly lower concentrations than ISO-1. Although all three MIF inhibitors inhibit caspase 3 activity, K664-1 and K647-1 suppress pro-apoptotic BAX protein expression and up-regulate anti-apoptotic Bcl-2 mRNA. Finally, all three MIF inhibitors operate through blockade of nitric oxide production stimulated by cytokines. In conclusion, two novel MIF inhibitors are more potent than ISO-1 and operate through inhibition of the mitochondria-related apoptotic pathway. We propose that these compounds represent a unique class of anti-MIF antagonists that should be further tested for therapeutic use.