RESUMEN
Small molecules derived from symbiotic microbiota critically contribute to intestinal immune maturation and regulation1. However, little is known about the molecular mechanisms that control immune development in the host-microbiota environment. Here, using a targeted lipidomic analysis and synthetic approach, we carried out a multifaceted investigation of immunomodulatory α-galactosylceramides from the human symbiont Bacteroides fragilis (BfaGCs). The characteristic terminal branching of BfaGCs is the result of incorporation of branched-chain amino acids taken up in the host gut by B. fragilis. A B. fragilis knockout strain that cannot metabolize branched-chain amino acids showed reduced branching in BfaGCs, and mice monocolonized with this mutant strain had impaired colonic natural killer T (NKT) cell regulation, implying structure-specific immunomodulatory activity. The sphinganine chain branching of BfaGCs is a critical determinant of NKT cell activation, which induces specific immunomodulatory gene expression signatures and effector functions. Co-crystal structure and affinity analyses of CD1d-BfaGC-NKT cell receptor complexes confirmed the interaction of BfaGCs as CD1d-restricted ligands. We present a structural and molecular-level paradigm of immunomodulatory control by interactions of endobiotic metabolites with diet, microbiota and the immune system.
Asunto(s)
Aminoácidos de Cadena Ramificada/inmunología , Aminoácidos de Cadena Ramificada/metabolismo , Bacteroides fragilis/metabolismo , Galactosilceramidas/inmunología , Galactosilceramidas/metabolismo , Microbioma Gastrointestinal/inmunología , Simbiosis/inmunología , Aminoácidos de Cadena Ramificada/química , Animales , Antígenos CD1d/inmunología , Bacteroides fragilis/genética , Humanos , Ratones , Modelos Animales , Modelos Moleculares , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunologíaRESUMEN
An antivirulence agent against Vibrio vulnificus named quoromycin (QM) was discovered by a phenotype-based elastase inhibitor screening. Using the fluorescence difference in two-dimensional gel electrophoresis (FITGE) approach, SmcR, a quorum-sensing master regulator and homologue of LuxR, was identified as the target protein of QM. We confirmed that the direct binding of QM to SmcR inhibits the quorum-sensing signaling pathway by controlling the DNA-binding affinity of SmcR and thus effectively alleviates the virulence of V. vulnificus in vitro and in vivo. QM can be regarded as a novel antivirulence agent for the treatment of V. vulnificus infection.
Asunto(s)
Vibrio vulnificus , Proteínas Bacterianas/genética , Fenotipo , Percepción de Quorum , Transactivadores/genéticaRESUMEN
α-Galactosylceramide (α-GalCer) is a typical antigen for invariant natural killer T cells that are a subset of T cells and play critical roles in regulating immune responses. To selectively induce the secretion of certain cytokines via introducing hydrogen-bonding interaction with polar amino acid residues in the binding pocket of CD1d, a series of α-GalCer analogues with diether moiety in the acyl chain were designed and synthesized. The subsequent in vitro biological evaluation of these analogues revealed the structure-activity relationship for the selective IL-17 secretion. Analogues 5 and 6 induced the significantly increased IL-17 secretion over other cytokines, suggesting protective effects against pathogens. In contrast, analogue 7 showed the highly reduced IL-17 secretion, which may indicate potential anti-inflammatory effects.
RESUMEN
A series of α-GalCer analogues containing an α-fluorocarbonyl moiety at the terminal position of the acyl chain were designed for targeting polar residues in the hydrophobic cavity of CD1d using a structure-based approach. The acyl chain length was efficiently adjusted by an asymmetric alkyne-alkyne cross coupling strategy, and the newly synthesized α-GalCer analogues showed the high Th2-selective activity of iNKT cells. The biased activity of ligands could be caused by the hydrogen-bonding interaction between ligands and CD1d according to the Th2-selective cytokine secretion and molecular docking studies.
RESUMEN
Microglial activation plays a pivotal role in the development and progression of neurodegenerative diseases. Thus, anti-inflammatory agents that control microglial activation can serve as potential therapeutic agents for neurodegenerative diseases. Here, we designed and synthesized α-galactosylceramide (α-GalCer) analogs to exert anti-inflammatory effects in activated microglia. We performed biological evaluations of 25 α-GalCer analogs and observed an interesting preliminary structure-activity relationship in their inhibitory influence on NO release and TNF-α production in LPS-stimulated BV2 microglial cells. After identification of 4d and 4e as hit compounds, we further investigated the underlying mechanism of their anti-inflammatory effects using RT-PCR analysis. We confirmed that 4d and 4e regulate the expression of iNOS, COX-2, IL-1ß, and IL-6 at the mRNA level and the expression of TNF-α at the post-transcriptional level. In addition, both 4d and 4e inhibited LPS-induced DNA binding activities of NF-κB and AP-1 and phosphorylation of p38 MAPK without affecting other MAP kinases. When we examined the anti-inflammatory effect of a p38 MAPK-specific inhibitor, SB203580, on microglial activation, we observed an identical inhibitory pattern as that of 4d and 4e, not only on NO and TNF-α production but also on the DNA binding activities of NF-κB and AP-1. Taken together, these results suggest that p38 MAPK plays an important role in the anti-inflammatory effects of 4d and 4e via the modulation of NF-κB and AP-1 activities.
Asunto(s)
Antiinflamatorios/química , Galactosilceramidas/química , Regulación Enzimológica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Microglía/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Supervivencia Celular , Células Cultivadas , Ciclooxigenasa 2/metabolismo , ADN/química , Imidazoles/química , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/química , Ratones , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/química , Piridinas/química , Factor de Transcripción AP-1/metabolismoRESUMEN
We designed and synthesized seven α-GalCer analogues with a pyrazole moiety and varying positions of a phenyl group in the sphingosine backbone to polarize cytokine secretion. On the basis of in vitro and in vivo biological evaluations, we found that analogue 5 induced greater polarization toward Th2 and greater secretion of the immunomodulatory cytokine, IL-4, over secretion of pro-inflammatory cytokines, IFN-γ and IL-17. Treatment of a single dose of analogue 5 markedly ameliorated disease pathogenesis in an animal model of an inflammatory demyelinating disease of the central nervous system, compared to that of KRN7000 (1). Therefore, this new α-GalCer analogue 5 is a novel iNKT ligand that stimulates the selective secretion of anti-inflammatory cytokines and regulates autoimmune diseases by reducing Th1 and Th17 responses.
Asunto(s)
Galactosilceramidas/síntesis química , Galactosilceramidas/farmacología , Esfingosina/química , Animales , Galactosilceramidas/química , Espectroscopía de Resonancia Magnética , Ratones , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
In this paper, we describe a regioselective synthetic pathway for enantiopure 1,3-disubstituted tetrahydroindazolone derivatives via the condensation of 2-acylcyclohexane-1,3-dione with various alkyl- and arylhydrazines using the steric effects of a Boc-protected pyrrolidine ring. This synthetic method has a broad scope for substrate generality for various hydrazines with excellent regioselectivity. To maximize the molecular diversity, further diversifications of 1,3-disubstituted tetrahydroindazolones were pursued by systematic N-modification of the secondary amine of the pyrrolidine ring using solution-phase parallel synthesis with polymer-supported reagents. A library containing a total of 272 drug-like tetrahydroindazolones, including 85 enantiomeric pairs, was constructed; the average purity, without further purification, was 95%.
Asunto(s)
Técnicas de Química Sintética/métodos , Descubrimiento de Drogas/métodos , Indazoles/síntesis química , Bibliotecas de Moléculas Pequeñas/síntesis química , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Células HeLa , Humanos , Indazoles/química , Indazoles/farmacología , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , EstereoisomerismoRESUMEN
We have developed a practical strategy for the regioselective synthesis of a 1-(hetero)aryl-3-substituted tetrahydroindazolone library. The condensation of in situ generated arylhydrazine on solid supports with 2-acylcyclohexane-1,3-diones ensured the efficiency of solid-phase parallel synthesis. In addition, we introduced three unique core skeletons containing nitrophenyl, anilyl, and pyridyl groups to maximize the molecular diversity through a diverse display of polar surface area in 3D chemical space. A 162-membered drug-like tetrahydroindazolone library was constructed in an average purity of 92% without further purification.