RESUMEN
Histone deacetylases (HDACs) are a class of enzymes that cleave acyl groups from lysine residues of histone and non-histone proteins. There are 18 human HDAC isoforms with different cellular targets and functions. Among them, HDAC6 was found to be overexpressed in different types of cancer. However, when used in monotherapy, HDAC6 inhibition by selective inhibitors fails to show pronounced anti-cancer effects. The HDAC6 enzyme also addresses non-histone proteins like α-tubulin and cortactin, making it important for cell migration and angiogenesis. Recently, the NLRP3 inflammasome was identified as an important regulator of inflammation and immune responses and, importantly, HDAC6 is critically involved the activation of the inflammasome. We herein report the design, synthesis and biological evaluation of a library of selective HDAC6 inhibitors. Starting from the previously published crystal structure of MAIP-032 in complex with CD2 of zHDAC6, we performed docking studies to evaluate additional possible interactions of the cap group with the L1-loop pocket. Based on the results we synthesized 13 novel HDAC6 inhibitors via the Groebke-Blackburn-Bienaymé three component reaction as the key step. Compounds 8k (HDAC1 IC50: 5.87 µM; HDAC6 IC50: 0.024 µM; selectivity factor (SF1/6): 245) and 8m (HDAC1 IC50: 3.07 µM; HDAC6 IC50: 0.026 µM; SF1/6: 118) emerged as the most potent and selective inhibitors of HDAC6 and outperformed the lead structure MAIP-032 (HDAC1 IC50: 2.20 µM; HDAC6 IC50: 0.058 µM; SF1/6: 38) both in terms of inhibitory potency and selectivity. Subsequent immunoblot analysis confirmed the high selectivity of 8k and 8m for HDAC6 in a cellular environment. While neither 8k and 8m nor the selectivity HDAC6 inhibitor tubastatin A showed antiproliferative effects in the U-87 MG glioblastoma cell line, compound 8m attenuated cell migration significantly in wound healing assays in U-87 MG cells. Moreover, in macrophages compounds 8k and 8m demonstrated significant inhibition of LPS-induced IL1B mRNA expression and TNF release. These findings suggest that our imidazo[1,2-a]pyridine-capped HDAC6 inhibitors may serve as promising candidates for the development of drugs to effectively treat NLRP3 inflammasome-driven inflammatory diseases.
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Proteína con Dominio Pirina 3 de la Familia NLR , Neoplasias , Humanos , Histona Desacetilasa 6 , Inflamasomas , Inhibidores de Histona Desacetilasas/química , Antiinflamatorios/farmacología , Neoplasias/tratamiento farmacológico , Línea Celular TumoralRESUMEN
Haemolytic disorders, such as sickle cell disease, are accompanied by the release of high amounts of labile heme into the intravascular compartment resulting in the induction of proinflammatory and prothrombotic complications in affected patients. In addition to the relevance of heme-regulated proteins from the complement and blood coagulation systems, activation of the TLR4 signalling pathway by heme was ascribed a crucial role in the progression of these pathological processes. Heme binding to the TLR4-MD2 complex has been proposed recently, however, essential mechanistic information of the processes at the molecular level, such as heme-binding kinetics, the heme-binding capacity and the respective heme-binding sites (HBMs) is still missing. We report the interaction of TLR4, MD2 and the TLR4-MD2 complex with heme and the consequences thereof by employing biochemical, spectroscopic, bioinformatic and physiologically relevant approaches. Heme binding occurs transiently through interaction with up to four HBMs in TLR4, two HBMs in MD2 and at least four HBMs in their complex. Functional studies highlight that mutations of individual HBMs in TLR4 preserve full receptor activation by heme, suggesting that heme interacts with TLR4 through different binding sites independently of MD2. Furthermore, we confirm and extend the major role of TLR4 for heme-mediated cytokine responses in human immune cells.
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Transducción de Señal , Receptor Toll-Like 4 , Humanos , Receptor Toll-Like 4/metabolismo , Sitios de Unión , Citocinas/metabolismo , Antígeno 96 de los Linfocitos/metabolismo , LipopolisacáridosRESUMEN
BACKGROUND: The purinergic receptor P2X7 plays a crucial role in infection, inflammation, and cell death. It is thought that P2X7 receptor stimulation triggers processing and release of the pro-inflammatory cytokine interleukin (IL)-1ß by activation of the NLRP3 inflammasome; however, the underlying mechanisms remain poorly understood. METHODS: Modulation of IL-1ß secretion was studied in THP-1 macrophages. Adenosine 5'-triphosphate (ATP), BzATP, nigericin and pharmacological inhibitors of P2X receptors, inflammatory caspases and the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome were used to characterize signaling. RESULTS: In primed macrophages, IL-1ß release was increased after P2X7 receptor activation by ATP and 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP). Pharmacological inhibition or genetic knockout of NLRP3 does not completely inhibit IL-1ß release in TLR2/1-primed macrophages. Increase in extracellular K+ as well as inhibition of caspase-1 or serine proteases maintained IL-1ß release in macrophages stimulated with P2X7 receptor agonists at 50%. CONCLUSIONS: Our findings suggest a previously unrecognized mechanism of P2X7 receptor mediated IL-1ß release and highlight the existence of an NLRP3-independent pathway in human macrophages. Video Abstract.
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Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Adenosina Trifosfato/metabolismo , Caspasa 1/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Receptores Purinérgicos P2X7/metabolismoRESUMEN
Sepsis is a life-threatening condition caused by the body's overwhelming response to an infection, such as pneumonia or urinary tract infection. It occurs when the immune system releases cytokines into the bloodstream, triggering widespread inflammation. If not treated, it can lead to organ failure and death. Unfortunately, sepsis has a high mortality rate, with studies reporting rates ranging from 20% to over 50%, depending on the severity and promptness of treatment. According to the World Health Organization (WHO), the annual death toll in the world is about 11 million. One of the main toxins responsible for inflammation induction are lipopolysaccharides (LPS, endotoxin) from Gram-negative bacteria, which rank among the most potent immunostimulants found in nature. Antibiotics are consistently prescribed as a part of anti-sepsis-therapy. However, antibiotic therapy (i) is increasingly ineffective due to resistance development and (ii) most antibiotics are unable to bind and neutralize LPS, a prerequisite to inhibit the interaction of endotoxin with its cellular receptor complex, namely Toll-like receptor 4 (TLR4)/MD-2, responsible for the intracellular cascade leading to pro-inflammatory cytokine secretion. The pandemic virus SARS-CoV-2 has infected hundreds of millions of humans worldwide since its emergence in 2019. The COVID-19 (Coronavirus disease-19) caused by this virus is associated with high lethality, particularly for elderly and immunocompromised people. As of August 2023, nearly 7 million deaths were reported worldwide due to this disease. According to some reported studies, upregulation of TLR4 and the subsequent inflammatory signaling detected in COVID-19 patients "mimics bacterial sepsis". Furthermore, the immune response to SARS-CoV-2 was described by others as "mirror image of sepsis". Similarly, the cytokine profile in sera from severe COVID-19 patients was very similar to those suffering from the acute respiratory distress syndrome (ARDS) and sepsis. Finally, the severe COVID-19 infection is frequently accompanied by bacterial co-infections, as well as by the presence of significant LPS concentrations. In the present review, we will analyze similarities and differences between COVID-19 and sepsis at the pathophysiological, epidemiological, and molecular levels.
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COVID-19 , Sepsis , Humanos , Anciano , SARS-CoV-2/metabolismo , Lipopolisacáridos , COVID-19/complicaciones , Receptor Toll-Like 4/metabolismo , Sepsis/metabolismo , Endotoxinas , Inflamación/complicaciones , Bacterias Gramnegativas/metabolismo , Citocinas/metabolismo , AntibacterianosRESUMEN
The nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome is an important regulator of inflammation and immune responses. Histone deacetylase 6 (HDAC6) has been implicated in the assembly and activation of the NLRP3 inflammasome in mouse cells, however, the role in human immune cells remains poorly understood. Here, we investigated the effect of HDAC6 deficiency on NLRP3-mediated interleukin (IL)-1ß release using proteolysis targeting chimeras (PROTAC) technology. We designed an HDAC6 PROTAC (A6) composed of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) and the E3 ligase ligand thalidomide and a control PROTAC (non-degrading control, nc-A6) that binds to HDAC6 but lacks the ability to induce HDAC6 degradation. A6 but not nc-A6 reduced HDAC6 levels in THP-1 macrophages without affecting cell viability. PROTAC A6 and nc-A6 significantly reduced the release of IL-1ß in a concentration-dependent manner, suggesting that HDAC6 deficiency is not necessary for inhibition of NLRP3 inflammasome-mediated IL-1ß release. We found that inhibition of the catalytic domain with HDAC inhibitor SAHA or the specific HDAC6 inhibitor tubastatin A is sufficient to reduce IL-1ß release indicating that the enzymatic activity of HDAC6 is critical for NLRP3 inflammasome function. Mechanistically, the observed effects of HDAC6 inhibition on NLRP3-mediated inflammatory responses could be attributed to its interaction with Toll-like receptor (TLR) signaling. Tubastatin A did not affect IL-1ß levels when added after TLR-mediated priming. Collectively, our findings indicate that HDAC6 inhibitors show potent anti-inflammatory activity and suppress IL-1ß release by human macrophages, independent of NLRP3 assembly and activation.
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Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Animales , Ratones , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Histona Desacetilasa 6/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Proteínas Portadoras/metabolismo , Receptores Toll-Like , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Caspasa 1/metabolismoRESUMEN
Aspidasept (Pep19-2.5) and its derivative Pep19-4LF ("Aspidasept II") are anti-infective and anti-inflammatory synthetic polypeptides currently in development for application against a variety of moderate to severe bacterial infections that could lead to systemic inflammation, as in the case of severe sepsis and septic shock, as well as application to non-systemic diseases in the case of skin and soft tissue infections (SSTI). In the present study, Aspidasept and Aspidasept II and their part structures were analysed with respect to their toxic behavior in different established models against a variety of relevant cells, and in electrophysiological experiments targeting the hERG channel according to ICH S7B. Furthermore, the effects in mouse models of neurobiological behavior and the local lymph node according to OECD test guideline 429 were investigated, as well as a rat model of repeated dose toxicology according to ICH M3. The data provide conclusive information about potential toxic effects, thus specifying a therapeutic window for the application of the peptides. Therefore, these data allow us to define Aspidasept concentrations for their use in clinical studies as parenteral application.
RESUMEN
Toll-like receptor 8 (TLR8) is an endosomal TLR that has an important role in the innate human immune system, which is involved in numerous pathological conditions. Excessive activation of TLR8 can lead to inflammatory and autoimmune diseases, which highlights the need for development of TLR8 modulators. However, only a few small-molecule modulators that selectively target TLR8 have been developed. Here, we report the synthesis and systematic investigation of the structure-activity relationships of a series of novel TLR8 negative modulators based on previously reported 6-(trifluoromethyl)pyrimidin-2-amine derivatives. Four compounds showed low-micromolar concentration-dependent inhibition of TLR8-mediated signaling in HEK293 cells. These data confirm that the 6-trifluoromethyl group and two other substituents on positions 2 and 4 are important structural elements of pyrimidine-based TLR8 modulators. Substitution of the main scaffold at position 2 with a methylsulfonyl group or para hydroxy/hydroxymethyl substituted benzylamine is essential for potent negative modulation of TLR8. Our best-in-class TLR8-selective modulator 53 with IC50 value of 6.2 µM represents a promising small-molecule chemical probe for further optimization to a lead compound with potent immunomodulatory properties.
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Receptor Toll-Like 8/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Estructura Molecular , Relación Estructura-Actividad , Receptor Toll-Like 8/inmunologíaRESUMEN
The polypeptide Pep19-2.5 (Aspidasept®) has been described to act efficiently against infection-inducing bacteria by binding and neutralizing their most potent toxins, i.e., lipopolysaccharides (LPS) and lipoproteins/peptides (LP), independent of the resistance status of the bacteria. The mode of action was described to consist of a primary Coulomb/polar interaction of the N-terminal region of Pep19-2.5 with the polar region of the toxins followed by a hydrophobic interaction of the C-terminal region of the peptide with the apolar moiety of the toxins. However, clinical development of Aspidasept as an anti-sepsis drug requires an in-depth characterization of the interaction of the peptide with the constituents of the human immune system and with other therapeutically relevant compounds such as antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs). In this contribution, relevant details of primary and secondary pharmacodynamics, off-site targets, and immunogenicity are presented, proving that Pep19-2.5 may be readily applied therapeutically against the deleterious effects of a severe bacterial infection.
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Antiinfecciosos/farmacología , Antiinflamatorios/farmacología , Endotoxemia/tratamiento farmacológico , Inflamación , Péptidos/farmacología , Animales , Antiinfecciosos/uso terapéutico , Antiinflamatorios/uso terapéutico , Modelos Animales de Enfermedad , Endotoxemia/inmunología , Humanos , Lipopolisacáridos , Ratones , Péptidos/uso terapéuticoRESUMEN
The intestinal epithelial barrier, together with the microbiome and local immune system, is a critical component that maintains intestinal homeostasis. Dysfunction may lead to chronic inflammation, as observed in inflammatory bowel diseases. Animal models have historically been used in preclinical research to identify and validate new drug targets in intestinal inflammatory diseases. Yet, limitations about their biological relevance to humans and advances in tissue engineering have forced the development of more complex three-dimensional reconstructed intestinal epithelium. By introducing immune and commensal microbial cells, these models more accurately mimic the gut's physiology and the pathophysiological changes occurring in vivo in the inflamed intestine. Specific advantages and limitations of two-dimensional (2D) and three-dimensional (3D) intestinal models such as coculture systems, organoids, and microfluidic devices to study inflammatory and immune-related responses are highlighted. While current cell culture models lack the cellular and molecular complexity observed in vivo, the emphasis is put on how these models can be used to improve preclinical drug development for inflammatory diseases of the intestine.
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Enfermedades Inflamatorias del Intestino , Mucosa Intestinal , Animales , Técnicas de Cocultivo , Desarrollo de Medicamentos , Humanos , Modelos Biológicos , OrganoidesRESUMEN
INTRODUCTION: Gram-negative bacterial infections represent still a severe problem of human health care, regarding the increase in multi-resistance against classical antibiotics and the lack of newly developed antimicrobials. For the fight against these germs, anti-infective agents must overcome and/or bind to the Gram-negative outer membrane consisting of a lipopolysaccharide (LPS, endotoxin) outer leaflet and an inner leaflet from phospholipids, with additional peripheral or integral membrane proteins (OMP's). AREAS COVERED: The current article reviews data of existing therapeutic options and summarizes newer approaches for targeting and neutralizing endotoxins, ranging from in vitro over in vivo animal data to clinical applications by using databases such as Medline. EXPERT OPINION: Conventional antibiotic treatment of the bacteria leads to their killing, but not necessary LPS neutralization, which may be a severe problem in particular for the systemic pathway. This is the reason why there is an increasing number of therapeutic approaches, which - besides combating whole bacteria - at the same time try to neutralize endotoxin within or outside the bacterial cells mainly responsible for the high inflammation induction in Gram-negative species.
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Antibacterianos/administración & dosificación , Bacterias Gramnegativas/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Animales , Antibacterianos/farmacología , Desarrollo de Medicamentos , Endotoxinas/antagonistas & inhibidores , Bacterias Gramnegativas/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/microbiología , Lipopolisacáridos/antagonistas & inhibidoresRESUMEN
Reports of tattoo-associated risks boosted the interest in tattoo pigment toxicity over the last decades. Nonetheless, the influence of tattoo pigments on skin homeostasis remains largely unknown. In vitro systems are not available to investigate the interactions between pigments and skin. Here, we established TatS, a reconstructed human full-thickness skin model with tattoo pigments incorporated into the dermis. We mixed the most frequently used tattoo pigments carbon black (0.02 mg/ml) and titanium dioxide (TiO2, 0.4 mg/ml) as well as the organic diazo compound Pigment Orange 13 (0.2 mg/ml) into the dermis. Tissue viability, morphology as well as cytokine release were used to characterize TatS. Effects of tattoo pigments were compared to monolayer cultures of human fibroblasts. The tissue architecture of TatS was comparable to native human skin. The epidermal layer was fully differentiated and the keratinocytes expressed occludin, filaggrin and e-cadherin. Staining of collagen IV confirmed the formation of the basement membrane. Tenascin C was expressed in the dermal layer of fibroblasts. Although transmission electron microscopy revealed the uptake of the tattoo pigments into fibroblasts, neither viability nor cytokine secretion was altered in TatS. In contrast, TiO2 significantly decreased cell viability and increased interleukin-8 release in fibroblast monolayers. In conclusion, TatS emulates healed tattooed human skin and underlines the advantages of 3D systems over traditional 2D cell culture in tattoo pigment research. TatS is the first skin model that enables to test the effects of pigments in the dermis upon tattooing.
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Colorantes/toxicidad , Dermis/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Tinta , Queratinocitos/efectos de los fármacos , Tatuaje/efectos adversos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Colorantes/metabolismo , Citocinas/metabolismo , Dermis/metabolismo , Dermis/ultraestructura , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Proteínas Filagrina , Humanos , Queratinocitos/metabolismo , Queratinocitos/ultraestructura , Hollín/toxicidad , Titanio/toxicidadRESUMEN
Toll-like receptors (TLRs) build the first barrier in the innate immune response and therefore represent promising targets for the modulation of inflammatory processes. Recently, the pyrogallol-containing TLR2 antagonists CU-CPT22 and MMG-11 were reported; however, their 1,2,3-triphenol motif renders them highly susceptible to oxidation and excludes them from use in extended experiments under aerobic conditions. Therefore, we have developed a set of novel TLR2 antagonists (1-9) based on the systematic variation of substructures, linker elements, and the hydrogen-bonding pattern of the pyrogallol precursors by using chemically robust building blocks. The novel series of chemically stable and synthetically accessible TLR2 antagonists (1-9) was pharmacologically characterized, and the potential binding modes of the active compounds were evaluated structurally. Our results provide new insights into structure-activity relationships and allow rationalization of structural binding characteristics. Moreover, they support the hypothesis that this class of TLR ligands bind solely to TLR2 and do not directly interact with TLR1 or TLR6 of the functional heterodimer. The most active compound from this series (6), is chemically stable, nontoxic, TLR2-selective, and shows a similar activity with regard to the pyrogallol starting points, thus indicating the variability of the hydrogen bonding pattern.
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Pirogalol/farmacología , Receptor Toll-Like 2/antagonistas & inhibidores , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Ligandos , Estructura Molecular , Pirogalol/síntesis química , Pirogalol/química , Relación Estructura-Actividad , Receptor Toll-Like 2/metabolismoRESUMEN
Toll-like receptor 2 (TLR2) and TLR8 are involved in the recognition of bacterial and viral components and are linked not only to protective antimicrobial immunity but also to inflammatory diseases. Recently, increasing attention has been paid to the receptor crosstalk between TLR2 and TLR8 to fine-tune innate immune responses. In this study, we report a novel dual TLR2/TLR8 antagonist, compound 24 that was developed by a modeling-guided synthesis approach. The modulator was optimized from the previously reported 1,3-benzothiazole derivative, compound 8. Compound 24 was pharmacologically characterized for the ability to inhibit TLR2- and TLR8-mediated responses in TLR-overexpressing reporter cells and THP-1 macrophages. The modulator showed high efficacy with IC50 values in the low micromolar range for both TLRs, selectivity towards other TLRs and low cytotoxicity. At TLR2, a slight predominance for the TLR2/1 heterodimer was found in reporter cells selectively expressing TLR2/1 or TLR2/6 heterodimers. Concentration ratio analysis in the presence of Pam3CSK4 or Pam2CSK4 indicated non-competitive antagonist behavior at hTLR2. In computational docking studies, a plausible alternative binding mode of compound 24 was predicted for both TLR2 and TLR8. Our results provide evidence that it is feasible to simultaneously and selectively target endosomal- and surface-located TLRs. We identified a small-molecule dual TLR2/8 antagonist that may serve as a valuable pharmacological tool to decipher the role of TLR2/8 co-signaling in inflammation.
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Benzotiazoles/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Receptor Toll-Like 2/antagonistas & inhibidores , Receptor Toll-Like 8/antagonistas & inhibidores , Benzotiazoles/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Interleucina-8/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Estructura Molecular , Multimerización de Proteína/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Células THP-1 , Receptor Toll-Like 2/química , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 8/química , Receptor Toll-Like 8/metabolismoRESUMEN
Interleukin (IL)-1 signaling leads to production of pro-inflammatory mediators and is regulated by receptor endocytosis. Lysosomotropic drugs have been linked to increased pro-inflammatory responses under sterile inflammatory conditions but the underlying mechanisms have not been fully elucidated. Here, we report that lysosomotropic drugs potentiate pro-inflammatory effects in response to IL-1ß via a mechanism involving reactive oxygen species, p38 mitogen-activated protein kinase and reduced IL-1 receptor internalization. Chloroquine and hydroxychloroquine increased IL-1ß-induced CXCL8 secretion in macrophages which was critically dependent on the lysosomotropic character and inhibition of macroautophagy but independent from the NLRP3 inflammasome. Co-stimulation with the autophagy inducer interferon gamma attenuated CXCL8 release. Other lysosomotropic drugs like bafilomycin A1, fluoxetine and chlorpromazine but also the endocytosis inhibitor dynasore showed similar pro-inflammatory responses. Increased cell surface expression of IL-1 receptor suggests reduced receptor degradation in the presence of lysosomotropic drugs. Our findings provide new insights into a potentially crucial immunoregulatory mechanism in macrophages that may explain how lysosomotropic drugs drive sterile inflammation.
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Autofagia/efectos de los fármacos , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Lisosomas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Receptores de Interleucina-1/antagonistas & inhibidores , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Cloroquina/farmacología , Clorpromazina/farmacología , Endocitosis/efectos de los fármacos , Fluoxetina/farmacología , Humanos , Inflamación/inmunología , Inflamación/patología , Lipopolisacáridos/farmacología , Lisosomas/inmunología , Macrófagos/inmunología , Transducción de Señal , Células THP-1RESUMEN
Toll-like receptor 2 (TLR2) forms heterodimers with either TLR1 or TLR6 to induce protective early inflammatory responses to pathogen- and damage-associated molecular patterns. However, excessive activation is associated with inflammatory and metabolic diseases. Several TLR2 antagonists have been described but pharmacological characterization is still at an early stage. Previously, we identified the potent and selective TLR2 antagonist MMG-11 by computational modelling and experimental validation. Here, we characterized the TLR2 antagonists MMG-11 and CU-CPT22 as well as the TIR-domain binding TLR2 antagonist C29 in TLR-overexpressing promoter cells as well as human and mouse macrophages. In line with our recent studies, MMG-11 abrogated pro-inflammatory cytokine secretion and NF-κB activation induced by different bacterial TLR2 agonists. MMG-11 preferentially inhibited TLR2/1 signaling in promoter cells stably expressing TLR2 heterodimers and mouse macrophages. Furthermore, the TLR2 antagonist blocked ligand-induced interaction of TLR2 with MyD88 and reduced MAP kinase and NF-κB activation. MMG-11 and CU-CPT22 but not C29 displaced Pam3CSK4 in an indirect binding assay confirming the competitive mode of action of MMG-11 and CU-CPT22. Isobologram analysis revealed additive and synergistic effects when the non-competitive antagonist C29 was combined with the competitive antagonist MMG-11 or CU-CPT22, respectively. In conclusion, we provide evidence that MMG-11 acts as a competitive antagonist with a predominance for the TLR2/1 heterodimer in human and mouse cells. Our results also indicate that MMG-11 is a model compound for studying TLR2 signaling.
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Macrófagos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Receptor Toll-Like 1/antagonistas & inhibidores , Receptor Toll-Like 2/antagonistas & inhibidores , Animales , Citocinas/metabolismo , Células HEK293 , Humanos , Lipopéptidos/química , Lipopéptidos/metabolismo , Lipopéptidos/farmacología , Macrófagos/metabolismo , Ratones , Unión Proteica , Multimerización de Proteína , Células RAW 264.7 , Bibliotecas de Moléculas Pequeñas/química , Células THP-1 , Receptor Toll-Like 1/química , Receptor Toll-Like 1/metabolismo , Receptor Toll-Like 2/química , Receptor Toll-Like 2/metabolismoRESUMEN
Oxidative stress and Th17 cytokines are important mediators of inflammation. Treatment with beta-adrenoceptor (ADRB) antagonists (beta-blockers) is associated with induction or aggravation of psoriasis-like skin inflammation, yet the underlying mechanisms are poorly understood. Herein, we identify lysosomotropic beta-blockers as critical inducers of IL23A in human monocyte-derived Langerhans-like cells under sterile-inflammatory conditions. Cytokine release was not mediated by cAMP, suggesting the involvement of ADRB-independent pathways. NFKB/NF-κB and MAPK14/p38 activation was required for propranolol-induced IL23A secretion whereas the NLRP3 inflammasome was dispensable. MAPK14 regulated recruitment of RELB to IL23A promoter regions. Without affecting the ubiquitin-proteasome pathway, propranolol increased lysosomal pH and induced a late-stage block in macroautophagy/autophagy. Propranolol specifically induced reactive oxygen species production, which was critical for IL23A secretion, in Langerhans-like cells. Our findings provide insight into a potentially crucial immunoregulatory mechanism in cutaneous dendritic cells that may explain how lysosomotropic drugs regulate inflammatory responses. ABBREVIATIONS: ATF: activating transcription factor; DC: dendritic cell; ChIP: chromatin immunoprecipitation; gDNA: genomic DNA; IL: interleukin; LAMP1: lysosomal associated membrane protein 1; LC: Langerhans cell; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MoDC: monocyte-derived DC; MoLC: monocyte-derived Langerhans-like cell; mtDNA: mitochondrial DNA; NAC: N-acetyl-L-cysteine; NLRP3: NLR family pyrin domain containing 3; PBMC: peripheral blood mononuclear cell; PI: propidium iodide; PYCARD/ASC: PYD and CARD domain containing; qRT-PCR: quantitative real-time PCR; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TLR: Toll-like receptor; TRAF6: TNF receptor associated factor 6; TNF: tumor necrosis factor; Ub: ubiquitin.
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Antagonistas Adrenérgicos beta/farmacología , Interleucina-23/biosíntesis , Células de Langerhans/metabolismo , Lisosomas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Autofagia/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Cloroquina/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamasomas/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-12/genética , Interleucina-12/metabolismo , Células de Langerhans/efectos de los fármacos , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Propranolol/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal , Células Th17/citología , Células Th17/efectos de los fármacos , Proteínas Ubiquitinadas/metabolismoRESUMEN
The endosomal Toll-like receptor 8 (TLR8) recognizes single-stranded RNA and initiates early inflammatory responses. Despite the importance of endosomal TLRs for human host defense against microbial pathogens, extensive activation may contribute to autoimmune and inflammatory diseases. In contrast to the recent progress made in the development of modulators of plasma membrane-bound TLRs, little is known about endosomal TLR modulation and very few TLR8 inhibitors have been reported. In this study, we discovered and validated novel small-molecule TLR8 inhibitors. Fourteen potential TLR8 modulators were experimentally validated in HEK293T cells stably overexpressing human TLR8 and THP-1 macrophages. Five compounds inhibited TLR8-mediated signaling, representing a hit rate of 36%. The three most potent compounds neither cause cellular toxicity nor inhibition of TLR signaling induced by other receptor subtypes. Conclusively, we experimentally confirm novel and selective, pyrimidine-based TLR8 inhibitors with low cytotoxicity that are relevant candidates for lead optimization and further mechanistic studies.
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Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Receptor Toll-Like 8/antagonistas & inhibidores , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Modelos Moleculares , Estructura Molecular , Pirimidinas/química , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Células THP-1 , Receptor Toll-Like 8/metabolismoRESUMEN
Outer membrane vesicles (OMVs) are secreted by Gram-negative bacteria and induce a stronger inflammatory response than pure LPS. After endocytosis of OMVs by macrophages, lipopolysaccharide (LPS) is released from early endosomes to activate its intracellular receptors followed by non-canonical inflammasome activation and pyroptosis, which are critically involved in sepsis development. Previously, we could show that the synthetic anti-endotoxin peptide Pep19-2.5 neutralizes inflammatory responses induced by intracellular LPS. Here, we aimed to investigate whether Pep19-2.5 is able to suppress cytoplasmic LPS-induced inflammation under more physiological conditions by using OMVs which naturally transfer LPS to the cytosol. Isothermal titration calorimetry revealed an exothermic reaction between Pep19-2.5 and Escherichia coli OMVs and the Limulus Amebocyte Lysate assay indicated a strong endotoxin blocking activity. In THP-1 macrophages and primary human macrophages Pep19-2.5 and polymyxin B reduced interleukin (IL)-1ß and tumor necrosis factor (TNF) release as well as pyroptosis induced by OMVs, while the Toll-like receptor 4 signaling inhibitor TAK-242 suppressed OMV-induced TNF and IL-1ß secretion, but not pyroptosis. Internalization of Pep19-2.5 was at least partially mediated by the P2X7 receptor in macrophages but not in monocytes. Additionally, a cell-dependent difference in the neutralization efficiency of Pep19-2.5 became evident in macrophages and monocytes, indicating a critical role for peptide-mediated IL-1ß secretion via the P2X7 receptor. In conclusion, we provide evidence that LPS-neutralizing peptides inhibit OMV-induced activation of the inflammasome/IL-1 axis and give new insights into the mechanism of peptide-mediated neutralization of cytoplasmic LPS suggesting an essential and cell-type specific role for the P2X7 receptor.
Asunto(s)
Antiinflamatorios/farmacología , Membrana Externa Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Lipopolisacáridos/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Péptidos/farmacología , Membrana Externa Bacteriana/inmunología , Línea Celular , Escherichia coli/inmunología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/microbiología , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Piroptosis/efectos de los fármacosRESUMEN
Antimicrobial peptides (AMPs) are in the focus of scientific research since the 1990s. In most cases, the main aim was laid on the design of AMP to kill bacteria effectively, with particular emphasis on broadband action and independency on antibiotic resistance. However, so far no approved drug on the basis of AMP has entered the market.Our approach of constructing AMP, called synthetic anti-lipopolysaccharide peptides (SALPs), on the basis of inhibiting the inflammatory action of lipopolysaccharide (LPS, endotoxin) from Gram-negative bacteria was focused on the neutralization of the decisive toxins. These are, beside LPS from Gram-negative bacteria, the lipoproteins (LP) from Gram-positive origin. Although some of the SALPs have an antibacterial action, the most important property is the high-affinity binding to LPS and LP, whether as constituent of the bacteria or in free form which prevents the damaging inflammation, that could otherwise lead to life-threatening septic shock. Most importantly, the SALP may inhibit inflammation independently of the resistance status of the bacteria, and so far the repeated use of the peptides apparently does not cause resistance of the attacking pathogens.In this chapter, an overview is given over the variety of possible applications in the field of fighting against severe bacterial infections, from the use in systemic infection/inflammation up to various topical applications such as anti-biofilm action and severe skin and soft tissue infections.
Asunto(s)
Antibacterianos/química , Moléculas de Patrón Molecular Asociado a Patógenos/antagonistas & inhibidores , Péptidos/química , Infecciones Bacterianas/tratamiento farmacológico , Endotoxinas , Bacterias Gramnegativas , Humanos , LipopolisacáridosRESUMEN
Lipopolysaccharide (LPS) sensing in the cytosol by the noncanonical inflammasome leads to pyroptosis and NLRP3 inflammasome activation. This mechanism may be more critical for sepsis development than recognition of LPS by Toll-like receptor 4. LPS is directly binding to its intracellular receptor caspase-4/5/11, mediated by outer membrane vesicles and guanylate-binding proteins that deliver LPS to the cytosol and mediate access of caspases to LPS. Caspase-11-dependent cleavage of gasdermin D is discussed as a link between LPS-induced activation of caspases and pyroptosis or NLRP3 inflammasome activation. Finally, we highlight recently described inhibitors of cytosolic LPS-triggered noncanonical inflammasome activation that might be considered as potential drugs for the treatment of sepsis.