RESUMO
T lymphocytes constitute a major effector cell population in autoimmune type 1 diabetes. Despite essential functions of mitochondria in regulating activation, proliferation, and apoptosis of T cells, little is known regarding T cell metabolism in the progression of human type 1 diabetes. In this study, we report, using two independent cohorts, that T cells from patients with type 1 diabetes exhibited mitochondrial inner-membrane hyperpolarization (MHP). Increased MHP was a general phenotype observed in T cell subsets irrespective of prior antigen exposure, and was not correlated with HbA1C levels, subject age, or duration of diabetes. Elevated T cell MHP was not detected in subjects with type 2 diabetes. T cell MHP was associated with increased activation-induced IFNγ production, and activation-induced IFNγ was linked to mitochondria-specific ROS production. T cells from subjects with type 1 diabetes also exhibited lower intracellular ATP levels. In conclusion, intrinsic mitochondrial dysfunction observed in type 1 diabetes alters mitochondrial ATP and IFNγ production; the latter is correlated with ROS generation. These changes impact T cell bioenergetics and function.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Mitocôndrias/metabolismo , Subpopulações de Linfócitos T/metabolismo , Trifosfato de Adenosina/metabolismo , Apoptose/imunologia , Biomarcadores , Carbocianinas/administração & dosagem , Diabetes Mellitus Tipo 1/imunologia , Glicólise , Humanos , Imunofenotipagem , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Potencial da Membrana Mitocondrial , Microscopia Confocal , Subpopulações de Linfócitos T/imunologiaRESUMO
Positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors can enhance ion channel currents and downstream effects of α7 stimulation. We investigated the approach of using noncompetitive antagonists to regulate α7 receptor function, potentially distinguishing effects requiring ion channel currents from signaling induced by nonconducting states. Three small readily reversible antagonists, (1S,2R,4R)-N,2,3,3-tetramethylbicyclo[2.2.1]heptan-2-amine (mecamylamine), N-(2.6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314), and 2-(dimethylamino)ethyl 4-(butylamino)benzoate (tetracaine), as well as three large slowly reversible antagonists, bis-(2,2,6,6-tetramethyl-4-piperidinyl) sebacate (BTMPS), 2,2,6,6-tetramethylpiperidin-4-yl heptanoate (TMPH), and 1,2,4,5-tetra-{5-[1-(3-benzyl)pyridinium]pent-1-yl}benzene tetrabromide (tkP3BzPB), were investigated for their effectiveness and voltage dependence in the inhibition of responses evoked by acetylcholine alone or augmented by the α7-selective PAM N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea (PNU-120596). Analyses of the small antagonists on PNU-120596-potentiated single-channel bursts indicated that each agent had a distinct mechanism of inhibition and only that of QX-314 was consistent with simple open channel block. In addition to decreasing channel open times and burst durations, mecamylamine and tetracaine induced unique subconductance states. To determine whether channel-blocking activity alone would be sufficient to prevent cell death, the antagonists were tested for their ability to protect α7-expressing cells from cytotoxic effects of the α7 agonist choline in combination with PNU-120596. Only tetracaine and tkP3BzPB, the two agents that had effects least consistent with simple ion channel block, were fully cytoprotective at concentrations that gave submaximal inhibition of macroscopic currents in oocytes. Further analyses indicated that toxicity produced by PNU-120596 and choline was calcium independent and likely an apoptotic event. Our results are consistent with the hypothesis that PAMs may modulate conformational states important for both channel activity and ion channel-independent signaling.
Assuntos
Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/fisiologia , Regulação Alostérica , Animais , Cálcio/metabolismo , Linhagem Celular , Colina/farmacologia , Citotoxinas/farmacologia , Feminino , Humanos , Isoxazóis/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Compostos de Fenilureia/farmacologia , Xenopus laevisRESUMO
α7 nicotinic acetylcholine receptors (nAChRs) have been a puzzle since their discovery in brain and non-neuronal tissues. Maximal transient probability of an α7 nAChR being open with rapid agonist applications is only 0.002. The concentration dependence of α7 responses measured from transfected cells and Xenopus laevis oocytes shows the same disparity in potency estimations for peak currents and net charge, despite being studied at 1000-fold different time scales. In both cases the EC50 was approximately 10-fold lower for net charge than for peak currents. The equivalence of the data obtained at such disparate time scales indicates that desensitization of α7 is nearly instantaneous. At high levels of agonist occupancy, the receptor is preferentially converted to a ligand-bound nonconducting state, which can be destabilized by the positive allosteric modulator N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea (PNU-120596). Such currents can be sufficiently large to be cytotoxic to the α7-expressing cells. Both the potentiating effect of PNU-120596 and the associated cytotoxicity have a high temperature dependence that can be compensated for by serum factors. Therefore, despite reduced potentiation at body temperatures, use of type II positive allosteric modulators may put cells that naturally express high levels of α7 nAChRs, such as neurons in the hippocampus and hypothalamus, at risk. With a low intrinsic open probability and high propensity toward the induction of nonconducting ligand-bound states, it is likely that the well documented regulation of signal transduction pathways by α7 nAChRs in cells such as those that regulate inflammation may be independent of ion channel activation and associated with the nonconducting conformational states.
Assuntos
Receptores Nicotínicos/fisiologia , Soroalbumina Bovina/farmacologia , Acetilcolina/farmacologia , Regulação Alostérica , Animais , Bovinos , Citotoxinas/farmacologia , Sinergismo Farmacológico , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoxazóis/farmacologia , Técnicas de Patch-Clamp , Compostos de Fenilureia/farmacologia , Ensaio Radioligante , Receptores Nicotínicos/genética , Temperatura , Transfecção , Receptor Nicotínico de Acetilcolina alfa7RESUMO
There is a pressing need for the development of novel, safe and effective adjuvants. The recent discovery and characterization of pathogen-associated molecular pattern (PAMP)-recognizing elements such as the Toll-like, NOD-like and RIG-like receptors, has brought into sharp focus the role of PAMPs in bridging the innate and adaptive immune responses, and a detailed understanding of the immunostimulatory vis-à-vis proinflammatory activities could lead to the development of effective adjuvants, monophosphoryl lipid A being an excellent example. We describe in this paper a series of hierarchical assays that were employed to characterize TLR agonists in vitro including primary TLR-reporter assays, secondary indices of immune activation, and tertiary screens characterizing transcriptomal activation patterns to identify optimal immunostimulatory chemotypes. The evaluation of representative members of known human TLR agonists demonstrate that TLR2, -4, -5 and -7 agonists were immunostimulatory. TLR7 agonists were extremely immunostimulatory, stimulating nearly all subsets of lymphocytes without inducing proinflammatory cytokine responses. The TLR5 agonist, flagellin, while immunostimulatory, was also highly proinflammatory. These results suggest that TLR agonists other than lipid A-like chemotypes could be developed into potential adjuvants, and that this series of hierarchical assays could be adapted to rapidly identify in large libraries, compounds with adjuvantic potential that lack proinflammatory responses.
Assuntos
Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/farmacologia , Sangue/imunologia , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologia , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Técnicas In Vitro , Ligantes , Ligação ProteicaRESUMO
The N-termini of bacterial lipoproteins are acylated with a (S)-(2,3-bisacyloxypropyl)cysteinyl residue. Lipopeptides derived from lipoproteins activate innate immune responses by engaging Toll-like receptor 2 (TLR2) and are highly immunostimulatory and yet without apparent toxicity in animal models. The lipopeptides may therefore be useful as potential immunotherapeutic agents. Previous structure-activity relationships in such lipopeptides have largely been obtained using murine cells, and it is now clear that significant species-specific differences exist between human and murine TLR responses. We have examined in detail the role of the highly conserved Cys residue as well as the geometry and stereochemistry of the Cys-Ser dipeptide unit. (R)-Diacylthioglycerol analogues are maximally active in reporter gene assays using human TLR2. The Cys-Ser dipeptide unit represents the minimal part-structure, but its stereochemistry was found not to be a critical determinant of activity. The thioether bridge between the diacyl and dipeptide units is crucial, and replacement by an oxoether bridge results in a dramatic decrease in activity.
Assuntos
Glicerol/análogos & derivados , Lipopeptídeos/química , Sulfetos/química , Receptor 2 Toll-Like/agonistas , Linhagem Celular , Cistina/química , Glicerol/química , Humanos , Lipopeptídeos/síntese química , Lipopeptídeos/farmacologia , NF-kappa B/biossíntese , Serina/química , Estereoisomerismo , Relação Estrutura-Atividade , Sulfetos/síntese química , Sulfetos/farmacologiaRESUMO
Amphotericin B (AmB), a well-known polyene antifungal agent, displays a marked tendency to self-associate and, as a consequence, exhibits very poor solubility in water. The therapeutic index of AmB is low and is associated with significant dose-related nephrotoxicity, as well as acute, infusion-related febrile reactions. Reports in the literature indicate that the toxicity of AmB may be related to the physical state of the drug. Reaction of AmB in dimethylformamide with bis(dimethylaminopropyl)carbodiimide yielded an unexpected N-alkylguanidine/N-acylurea bis-adduct of AmB which was highly water-soluble. The absorption spectrum of the AmB derivative in water indicated excellent monomerization, and the antifungal activities of reference AmB and its water-soluble derivative against Candida albicans were found to be virtually identical. Furthermore, the water-soluble adduct is significantly less active in engaging TLR4, which would suggest that the adduct may be less proinflammatory.
Assuntos
Anfotericina B/análogos & derivados , Antifúngicos/síntese química , Antifúngicos/toxicidade , Anfotericina B/síntese química , Anfotericina B/toxicidade , Antifúngicos/química , Candida albicans/efeitos dos fármacos , Linhagem Celular , Genes Reporter , Humanos , Inflamação/induzido quimicamente , Rim/efeitos dos fármacos , Rim/metabolismo , Estrutura Molecular , Solubilidade , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , ÁguaRESUMO
Toll-like receptors (TLRs) are a family of conserved pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns and serve as primary sensors of the innate immune system. Ten members of the TLR family have so far been identified in the human genome. The ligands for these receptors are structurally highly conserved microbial molecules such as lipopolysaccharides (LPS) (recognized by TLR4), lipopeptides (TLR2 in combination with TLR1 or TLR6), flagellin (TLR5), single stranded RNA (TLR7 and TLR8), double-stranded RNA (TLR3), CpG motif-containing DNA (TLR9) and profilin present on uropathogenic bacteria (TLR 11). Complementing the TLRs are the nucleotide-binding domain (NOD), leucine rich repeat containing family (or Nod-like Receptors, NLRs), which detect muramylpeptides released from bacterial peptidoglycan (PGN) in the intracytoplasmic compartment, as well as the retinoic-acid-inducible protein 1 (RIG-I-like receptors; RLRs) which sense single-stranded RNA of viral origin. The activation of PRRs by their cognate ligands leads to production of inflammatory cytokines, upregulation of MHC molecules and co-stimulatory signals in antigen-presenting cells as well as activating natural killer cells, in addition to priming and amplifying antigen-specific T-, and B-cell effector functions. Thus, these stimuli serve to link innate and adaptive immunity and can therefore be exploited as powerful adjuvants in eliciting both primary and anamnestic immune responses. This review summarizes what is currently known about the immunopotentiatory and adjuvantic activities of innate immune stimuli.
Assuntos
Adjuvantes Imunológicos/farmacologia , Imunidade Inata , Receptores Imunológicos/agonistas , HumanosRESUMO
Chronic immune activation is a hallmark of progressive HIV infection. Recent reports point to the engagement of toll-like receptor 7 (TLR7) and -9 by viral RNA as contributing to the activation of innate immune responses, which drive viral replication leading to immune exhaustion. The only known class of TLR7 antagonists is single-stranded phosphorothioate oligonucleotides, which has been demonstrated to inhibit immune activation in human and Rhesus macaque in vitro models. The availability of a selective and potent small-molecule TLR7 antagonist should allow the evaluation of potential benefits of suppression of TLR7-mediated immune activation in HIV/AIDS. Gardiquimod is a known N(1)-substituted 1H-imidazoquinoline TLR7 agonist, the synthesis of which has not been published. We show that the 3H regioisomer is completely inactive as a TLR7 agonist and is weakly antagonistic. A des-amino precursor of the 3H regioisomer is more potent as a TLR7 antagonist, with an IC(50) value of 7.5 microM. This class of compound may serve as a starting point for the development of small-molecule inhibitors of TLR7.
Assuntos
Imidazóis/química , Imidazóis/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/antagonistas & inibidores , Linhagem Celular , Cristalografia por Raios X , Humanos , Estereoisomerismo , Receptor 7 Toll-Like/fisiologiaRESUMO
The toxicity of gram-negative bacterial endotoxin (lipopolysaccharide, LPS) resides in its structurally highly conserved glycolipid component called lipid A. Our major goal has been to develop small-molecules that would sequester LPS by binding to the lipid A moiety, so that it could be useful for the prophylaxis or adjunctive therapy of gram-negative sepsis. We had previously identified in rapid-throughput screens several guanylhydrazones as potent LPS binders. We were desirous of examining if the presence of the guanylhydrazone (rather than an amine) functionality would afford greater LPS sequestration potency. In evaluating a congeneric set of guanylhydrazone analogues, we find that C(16) alkyl substitution is optimal in the N-alkylguanylhydrazone series; a homospermine analogue with the terminal amine N-alkylated with a C(16) chain with the other terminus of the molecule bearing an unsubstituted guanylhydrazone moiety is marginally more active, suggesting very slight, if any, steric effects. Neither C(16) analogue is significantly more active than the N-C(16)-alkyl or N-C(16)-acyl compounds that we had characterized earlier, indicating that basicity of the phosphate-recognizing cationic group, is not a determinant of LPS sequestration activity.
Assuntos
Antibacterianos/síntese química , Hidrazonas/química , Lipopolissacarídeos/metabolismo , Poliaminas/química , Antibacterianos/farmacologia , Sítios de Ligação , Bactérias Gram-Negativas , Lipídeo A/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Poliaminas/farmacologia , Relação Estrutura-AtividadeRESUMO
The role of lipopolysaccharide (LPS) in the pathogenesis of Gram-negative septic shock is well established. The corresponding proinflammatory and immunostimulatory molecule(s) on the Gram-positive bacteria is less well understood, and its identification and characterization would be a key prerequisite in designing specific sequestrants of the Gram-positive endotoxin(s). We report in this paper the comparison of NF-kappaB-, cytokine- and chemokine-inducing activities of the TLR2 ligands, lipoteichoic acid (LTA), peptidoglycan (PGN), and lipopeptides, to LPS, a prototype TLR4 agonist, in murine macrophage cell-lines as well as in human blood. In murine cells, di- and triacyl liopopeptides are equipotent in their NF-kappaB inducing activity relative to LPS, but elicit much lower proinflammatory cytokines. However, both LPS and the lipopeptides potently induce the secretion of a pattern of chemokines that is suggestive of the engagement of a TLR4-independent TRIF pathway. In human blood, although the lipopeptides induce p38 MAP kinase phosphorylation and CD11b upregulation in granulocytes at ng/ml concentrations, they do not elicit proinflammatory cytokine production even at very high doses; LTA, however, activates neutrophils and induces cytokine secretion, although its potency is considerably lower than that of LPS, presumably due to its binding to plasma proteins. We conclude that, in human blood, the pattern of immunostimulation and proinflammatory mediator production elicited by LTA parallels that of LPS.
Assuntos
Adjuvantes Imunológicos/farmacologia , Células/efeitos dos fármacos , Endotoxinas/farmacologia , Inflamação/induzido quimicamente , Lipopolissacarídeos/farmacologia , Lipoproteínas/farmacologia , Peptidoglicano/farmacologia , Ácidos Teicoicos/farmacologia , Animais , Células Sanguíneas/efeitos dos fármacos , Linhagem Celular , Endotoxinas/imunologia , Bactérias Gram-Positivas/química , Humanos , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Lipoproteínas/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Peptidoglicano/imunologia , Ácidos Teicoicos/imunologia , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/sangue , Receptor 2 Toll-Like/metabolismoRESUMO
Lipopolysaccharides (LPS) play a key role in the pathogenesis of septic shock, a major cause of mortality in the critically ill patient. The only therapeutic option aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide is Toraymyxin, an extracorporeal hemoperfusion device using solid phase-immobilized polymyxin B (PMB). While PMB is known to effectively sequester LPS, its severe systemic toxicity proscribes its parenteral use, and hemoperfusion may not be feasible in patients in shock. In our continuing efforts to develop small-molecule mimics which display the LPS-sequestering properties, but not the toxicity of PMB, a series of mono- and bis-substituted dialkylpolyamines were synthesized and evaluated. We show that EVK-203, an alkylpolyamine compound, specifically binds to and neutralizes the activity of LPS, and affords complete protection in a murine model of endotoxic shock. EVK-203 is without any apparent toxicity when administered to mice at multiples of therapeutic doses for several days. The specific endotoxin-sequestering property along with a very favorable therapeutic index renders this compound an ideal candidate for preclinical development.
Assuntos
Citoproteção/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Poliaminas/química , Poliaminas/farmacologia , Choque Séptico/tratamento farmacológico , Animais , Linhagem Celular , Citocinas/sangue , Modelos Animais de Doenças , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Poliaminas/síntese química , Choque Séptico/sangue , Choque Séptico/induzido quimicamente , Choque Séptico/patologia , Relação Estrutura-Atividade , Testes de ToxicidadeRESUMO
Lipopolysaccharide (LPS), or endotoxin, a structural component of gram-negative bacterial outer membranes, plays a key role in the pathogenesis of septic shock, a syndrome of severe systemic inflammation which leads to multiple-system organ failure. Despite advances in antimicrobial chemotherapy, sepsis continues to be the commonest cause of death in the critically ill patient. This is attributable to the lack of therapeutic options that aim at limiting the exposure to the toxin and the prevention of subsequent downstream inflammatory processes. Polymyxin B (PMB), a peptide antibiotic, is a prototype small molecule that binds and neutralizes LPS toxicity. However, the antibiotic is too toxic for systemic use as an LPS sequestrant. Based on a nuclear magnetic resonance-derived model of polymyxin B-LPS complex, we had earlier identified the pharmacophore necessary for optimal recognition and neutralization of the toxin. Iterative cycles of pharmacophore-based ligand design and evaluation have yielded a synthetically easily accessible N(1),mono-alkyl-mono-homologated spermine derivative, DS-96. We have found that DS-96 binds LPS and neutralizes its toxicity with a potency indistinguishable from that of PMB in a wide range of in vitro assays, affords complete protection in a murine model of LPS-induced lethality, and is apparently nontoxic in vertebrate animal models.
Assuntos
Antibacterianos , Endotoxinas/química , Lipopolissacarídeos/química , Choque Séptico/prevenção & controle , Espermina , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Linhagem Celular , Endotoxinas/toxicidade , Feminino , Humanos , Lipopolissacarídeos/toxicidade , Macrófagos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Polimixina B/química , Polimixina B/uso terapêutico , Espermina/análogos & derivados , Espermina/química , Espermina/farmacologia , Espermina/uso terapêutico , Resultado do TratamentoRESUMO
Lipopolysaccharides (LPS) play a key role in the pathogenesis of septic shock, a major cause of mortality in the critically ill patient. We had previously shown that monoacylated polyamine compounds specifically bind to and neutralize the activity of LPS with high in vitro potency and afford complete protection in a murine model of endotoxic shock. Fatty acid amides of polyamines may be rapidly cleared from systemic circulation due to their susceptibility to nonspecific serum amidases and, thus, would be predicted to have a short duration of action. In a systematic effort to increase the likelihood of better bioavailability properties together with structural modifications that may result in gains in activity, we now report structure-activity relationships pertaining to endotoxin-binding and -neutralizing activities of homologated polyamine sulfonamides.
Assuntos
Lipopolissacarídeos/metabolismo , Espermina/análogos & derivados , Espermina/síntese química , Sulfonamidas/síntese química , Animais , Cátions , Citocinas/antagonistas & inibidores , Citocinas/sangue , Feminino , Humanos , Técnicas In Vitro , Lipopolissacarídeos/intoxicação , Camundongos , NF-kappa B/antagonistas & inibidores , Óxido Nítrico/antagonistas & inibidores , Espermina/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/farmacologiaRESUMO
Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria, and play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient. We had previously defined the pharmacophore necessary for small molecules to specifically bind and neutralize this complex carbohydrate. A series of aryl and aliphatic spermine-sulfonamide analogs were synthesized and tested in a series of binding and cell-based assays in order to probe the effect of lipophilicity on sequestration ability. A strong correlation was indeed found, supporting the hypothesis that endotoxin-neutralizing ability involves a lipophilic or membrane attachment event. The research discussed herein may be useful for the design of additional carbohydrate recognizing molecules and endotoxin-neutralizing drugs.
Assuntos
Lipopolissacarídeos/química , Lipopolissacarídeos/isolamento & purificação , Espermina/análogos & derivados , Espermina/química , Sulfonamidas/química , Configuração de Carboidratos , Cinética , Modelos Moleculares , Espermina/farmacocinética , Relação Estrutura-Atividade , Sulfonamidas/farmacocinéticaRESUMO
We have shown that lipopolyamines bind to the lipid A moiety of lipopolysaccharide, a constituent of Gram-negative bacterial membranes, and neutralize its toxicity in animal models of endotoxic shock. In an effort to identify non-polyamine scaffolds with similar endotoxin-recognizing features, we had observed an unusually high frequency of hits containing guanylhydrazone scaffolds in high-throughput screens. We now describe the syntheses and preliminary structure-activity relationships in a homologous series of bis-guanylhydrazone compounds decorated with hydrophobic functionalities. These first-generation compounds bind and neutralize lipopolysaccharide with a potency comparable to that of polymyxin B, a peptide antibiotic known to sequester LPS.