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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Here we identify a group 2 innate lymphoid cell (ILC2) subpopulation that can convert into interleukin-17 (IL-17)-producing NKp44- ILC3-like cells. c-Kit and CCR6 define this ILC2 subpopulation that exhibits ILC3 features, including RORγt, enabling the conversion into IL-17-producing cells in response to IL-1ß and IL-23. We also report a role for transforming growth factor-ß in promoting the conversion of c-Kit- ILC2s into RORγt-expressing cells by inducing the upregulation of IL23R, CCR6 and KIT messenger RNA in these cells. This switch was dependent on RORγt and the downregulation of GATA-3. IL-4 was able to reverse this event, supporting a role for this cytokine in maintaining ILC2 identity. Notably, this plasticity has physiological relevance because a subset of RORγt+ ILC2s express the skin-homing receptor CCR10, and the frequencies of IL-17-producing ILC3s are increased at the expense of ILC2s within the lesional skin of patients with psoriasis.
Assuntos
Interleucina-17/imunologia , Linfócitos/imunologia , Psoríase/patologia , Pele/patologia , Células Cultivadas , Humanos , Interleucina-1beta/imunologia , Subunidade p19 da Interleucina-23/imunologia , Interleucina-4/imunologia , Linfócitos/citologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Psoríase/imunologia , Receptores CCR10/metabolismo , Pele/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Full activation of naive T cells requires both engagement of the T cell antigen receptor (TCR; signal 1) and costimulatory signaling by CD28 (signal 2). We previously identified two types of rat CD28-specific monoclonal antibodies (mAbs): "conventional," TCR signaling-dependent costimulatory mAbs and "superagonistic" mAbs capable of inducing the full activation of primary resting T cells in the absence of TCR ligation both in vitro and in vivo. Using chimeric rat/mouse CD28 molecules, we show that the superagonists bind exclusively to the laterally exposed C"D loop of the immunoglobulin-like domain of CD28 whereas conventional, costimulatory mAbs recognize an epitope close to the binding site for the natural CD80/CD86 ligands. Unexpectedly, the C"D loop reactivity of a panel of new antibodies raised against human CD28 could be predicted solely on the basis of their superagonistic properties. Moreover, mouse CD28 molecules engineered to express the rat or human C"D loop sequences activated T cell hybridomas without TCR ligation when cross-linked by superagonistic mAbs. Finally, biochemical analysis revealed that superagonistic CD28 signaling activates the nuclear factor kappaB pathway without inducing phosphorylation of either TCRzeta or ZAP70. Our findings indicate that the topologically constrained interactions of anti-CD28 superagonists bypass the requirement for signal 1 in T cell activation. Antibodies with this property may prove useful for the development of T cell stimulatory drugs.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos CD28/imunologia , Ativação Linfocitária , Conformação Proteica , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Antígenos CD28/genética , Antígenos CD28/metabolismo , Epitopos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , NF-kappa B/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70RESUMO
Members of the IL-6/IL-12 cytokine family are critical regulators of innate and adaptive immunity and have emerged as key players controlling inflammatory and autoimmune disorders. This cytokine family comprises of IL-12, IL-23, IL-27, and IL-35, each consisting of distinct α- and ß-cytokine subunits that form heterodimers. A new member of this family, IL-39, was identified in the murine species and was shown to consist of the IL-23p19 and Epstein-Barr Virus-induced 3 (EBI3) subunits. Subsequently, it was shown that IL-39 was implicated in the immunopathogenesis of murine experimental lupus erythematosus. The existence of IL-39 in the human system has yet to be confirmed. Based on the clinical success of IL-23p19 neutralizing approaches in moderate-to-severe psoriasis, anti-IL-23p19 antibodies in the clinic may not only neutralize IL-23, but additionally IL-39, implying that IL-39 might also contribute to the pathogenesis of psoriasis. It is therefore pivotal to demonstrate IL-39 expression and to characterize its function in the human system. In this study, we provided evidence for the existence of secreted heterodimeric p19 and EBI3 complexes in supernatants originating from p19 and EBI3 transfected HEK293FT cells. We attempted to detect IL-39 expression from stimulated human primary B cells, human keratinocytes and in vitro polarized human macrophages. Whereas, the expression of p19 and EBI3 mRNA was elevated, we failed to detect p19 and EBI3 heterodimers. Functional assays were conducted with conditioned media containing human IL-39 or with a human recombinant IL-39 Fc protein. Immune cells targeted by IL-39 in mouse, such as neutrophils and PBMCs, did not respond to human IL-39 stimulation and IL-39 failed to activate STAT3 in a reporter cell line. These results suggest that, while the secretion of p19/EBI3 complexes can be forced in human cells, it is secreted below the lower quantity of detection or it has no functional role.
Assuntos
Imunidade Adaptativa/genética , Imunidade Inata/genética , Subunidade p19 da Interleucina-23/genética , Interleucinas/genética , Antígenos de Histocompatibilidade Menor/genética , Receptores de Citocinas/genética , Animais , Humanos , Subunidade p19 da Interleucina-23/imunologia , Interleucinas/imunologia , Camundongos , Antígenos de Histocompatibilidade Menor/imunologia , Neutrófilos/imunologia , Receptores de Citocinas/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologiaRESUMO
There is a pressing need for immunosuppressants with an improved safety profile. The search for novel approaches to blocking T-cell activation led to the development of the selective protein kinase C (PKC) inhibitor AEB071 (sotrastaurin). In cell-free kinase assays AEB071 inhibited PKC, with K(i) values in the subnanomolar to low nanomolar range. Upon T-cell stimulation, AEB071 markedly inhibited in situ PKC catalytic activity and selectively affected both the canonical nuclear factor-kappaB and nuclear factor of activated T cells (but not activator protein-1) transactivation pathways. In primary human and mouse T cells, AEB071 treatment effectively abrogated at low nanomolar concentration markers of early T-cell activation, such as interleukin-2 secretion and CD25 expression. Accordingly, the CD3/CD28 antibody- and alloantigen-induced T-cell proliferation responses were potently inhibited by AEB071 in the absence of nonspecific antiproliferative effects. Unlike former PKC inhibitors, AEB071 did not enhance apoptosis of murine T-cell blasts in a model of activation-induced cell death. Furthermore, AEB071 markedly inhibited lymphocyte function-associated antigen-1-mediated T-cell adhesion at nanomolar concentrations. The mode of action of AEB071 is different from that of calcineurin inhibitors, and AEB071 and cyclosporine A seem to have complementary effects on T-cell signaling pathways.
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Imunossupressores/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirróis/antagonistas & inibidores , Quinazolinas/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Animais , Antígenos CD28/metabolismo , Cálcio/metabolismo , Adesão Celular/efeitos dos fármacos , Citocinas/biossíntese , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Genes Reporter , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Células Jurkat , Camundongos , Camundongos Knockout , NF-kappa B/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Proteína Quinase C/genética , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
The nuclear hormone receptor retinoic acid receptor-related-orphan-receptor-gamma t (RORγt) is the key transcription factor required for Th17 cell differentiation and for production of IL-17 family cytokines by innate and adaptive immune cells. Dysregulated Th17 immune responses have been associated with the pathogenesis of several inflammatory and autoimmune diseases such as psoriasis, psoriatic arthritis, and ankylosing spondylitis. In this article, we describe the in vitro pharmacology of a potent and selective low molecular weight RORγt inhibitor identified after a structure-based hit-to-lead optimization effort. The compound interfered with co-activator binding to the RORγt ligand binding domain and impaired the transcriptional activity of RORγt as evidenced by blocked IL-17A secretion and RORE-mediated transactivation of a luciferase reporter gene. The inhibitor effectively reduced IL-17A production by human naive and memory T-cells and attenuated transcription of pro-inflammatory Th17 signature genes, such as IL17F, IL22, IL26, IL23R, and CCR6. The compound selectively suppressed the Th17/IL-17 pathway and did not interfere with polarization of other T helper cell lineages. Furthermore, the inhibitor was selective for RORγt and did not modify the transcriptional activity of the closely related family members RORα and RORß. Using human keratinocytes cultured with supernatants from compound treated Th17 cells we showed that pharmacological inhibition of RORγt translated to suppressed IL-17-regulated gene expression in keratinocyte cell cultures. Furthermore, in ex vivo immersion skin cultures our RORγt inhibitor suppressed IL-17A production by Th17-skewed skin resident cells which correlated with reduced human ß defensin 2 expression in the skin. Our data suggests that inhibiting RORγt transcriptional activity by a low molecular weight inhibitor may hold utility for the treatment of Th17/IL-17-mediated skin pathologies.
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Interleucina-17/fisiologia , Queratinócitos/imunologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Pele/patologia , Células Th17/fisiologia , Acetatos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/fisiologia , Fator de Transcrição STAT3/fisiologia , Transdução de Sinais/fisiologia , Células Th17/citologia , Tiramina/análogos & derivados , Tiramina/farmacologiaRESUMO
Retinoic acid receptor-related orphan receptor gamma-t (RORγt) is considered to be the master transcription factor for the development of Th17 cells that produce proinflammatory cytokines such as IL-17A. Overproportionate Th17 cell abundance is associated with the pathogenesis of many inflammatory conditions including psoriasis. In a high-throughput fluorescence resonance energy transfer (FRET) screen, we identified compound 1 as a hit with promising lipophilic efficiency (LipE). Using structure-based drug design based on a number of X-ray cocrystal structures, we morphed this hit class into potent imidazoles, exemplified by compound 3. To improve the poor absorption, distribution, metabolism, and excretion (ADME) properties of neutral imidazoles, we extended our ligands with carboxylic acid substituents toward a polar, water-rich area of the protein. This highly lipophilicity-efficient modification ultimately led to the discovery of compound 14, a potent and selective inhibitor of RORγt with good ADME properties and excellent in vivo pharmacokinetics. This compound showed good efficacy in an in vivo delayed-type hypersensitivity pharmacology model in rats.
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Hipersensibilidade Tardia/tratamento farmacológico , Imidazóis/farmacologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Administração Oral , Animais , Relação Dose-Resposta a Droga , Desenho de Fármacos , Feminino , Transferência Ressonante de Energia de Fluorescência , Meia-Vida , Imidazóis/química , Imidazóis/farmacocinética , Masculino , Modelos Moleculares , Estrutura Molecular , RatosRESUMO
A large body of data demonstrates that interferon regulatory factor 5 (IRF5) and nuclear factor kappa B (NF-κB) are the two major transcription factors in classically activated macrophages responsible for the transcriptional control of proinflammatory genes. Although recent evidence suggests that IRF5 interacts with certain members of the nuclear factor kappa B pathway, the extent of cooperation and its implications in disease are ambiguous. Since both pathways are known for their strong contributions in TLR8 signaling we used the human monocytic cell line THP-1.Dual, featuring gene reporters for NF-κB and IRFs, to simultaneously study the roles of IRF5 and the NF-κB subunit p65 in TLR8-mediated gene reporter activities. Furthermore, we profiled from these cells the proinflammatory cytokines involved in the differentiation of TH1 and TH17 cells. After ablation of IRF5 and/or p65 we activated the resultant cells with the TLR8 agonists R848 or the psoriasis-associated antimicrobial peptide LL-37 complexed with ssRNA and demonstrate that IRF5 deficiency drastically impairs the secretion of IL-1ß, IL-6, IL-12, IL-23 and TNFα. In contrast, the lack of p65 impaired only IL-6, IL-12, and IL-23 secretion. Furthermore, we discovered that upon TLR8 stimulation, IRF5 but not NF-κB signaling is essential to provide a cytokine milieu supporting TH1 responses. Additionally, we demonstrate that IRF5 and NF-κB cooperate to provide a cytokine milieu supporting TH17 responses. Therefore, the distinct role of IRF5 in the intricate signaling network downstream of TLR8 may open new treatment options interfering with but not disrupting NF-κB signaling in human diseases.
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Inflamação/metabolismo , Fatores Reguladores de Interferon/metabolismo , NF-kappa B/metabolismo , Células Th1/imunologia , Células Th17/imunologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Citocinas/metabolismo , Técnicas de Inativação de Genes , Genes Reporter , Humanos , Imidazóis/farmacologia , Fatores Reguladores de Interferon/genética , NF-kappa B/genética , Elementos de Resposta , Transdução de Sinais , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th17/efeitos dos fármacos , Células Th17/metabolismo , Receptor 8 Toll-Like/agonistas , Receptor 8 Toll-Like/imunologia , Receptor 8 Toll-Like/metabolismo , CatelicidinasRESUMO
The transcription factor RORγt is an attractive drug-target due to its role in the differentiation of IL-17 producing Th17 cells that play a critical role in the etiopathology of several autoimmune diseases. Identification of starting points for RORγt inverse agonists with good properties has been a challenge. We report the identification of a fragment hit and its conversion into a potent inverse agonist through fragment optimization, growing and merging efforts. Further analysis of the binding mode revealed that inverse agonism was achieved by an unusual mechanism. In contrast to other reported inverse agonists, there is no direct interaction or displacement of helix 12 observed in the crystal structure. Nevertheless, compound 9 proved to be efficacious in a delayed-type hypersensitivity (DTH) inflammation model in rats.
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Descoberta de Drogas , Agonismo Inverso de Drogas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Animais , Domínio Catalítico , Modelos Animais de Doenças , Feminino , Inflamação/metabolismo , Modelos Moleculares , RatosRESUMO
The chemokine receptors CXCR4 and CCR5 are required for HIV-1 to enter cells, and the progression of HIV-1 infection to AIDS involves a switch in the co-receptor usage of the virus from CCR5 to CXCR4. These receptors therefore make attractive candidates for therapeutic intervention, and we have investigated the silencing of their genes by using ribozymes and single-stranded antisense RNAs. In the present study, we demonstrate using ribozymes that a depletion of CXCR4 and CCR5 mRNAs can be achieved simultaneously in human PBMCs (peripheral blood mononuclear cells), cells commonly used by the virus for infection and replication. Ribozyme activity leads to an inhibition of the cell-surface expression of both CCR5 and CXCR4, resulting in a significant inhibition of HIV-1 replication when PBMCs are challenged with the virus. In addition, we show that small single-stranded antisense RNAs can also be used to silence CCR5 and CXCR4 genes when delivered to PBMCs. This silencing is caused by selective degradation of receptor mRNAs.
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Inativação Gênica , HIV-1/fisiologia , RNA Antissenso/genética , RNA Antissenso/metabolismo , RNA Catalítico/genética , RNA Catalítico/metabolismo , Receptores CCR5/genética , Receptores CXCR4/genética , Células Cultivadas , RNA Polimerases Dirigidas por DNA/metabolismo , Citometria de Fluxo , Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , RNA Antissenso/biossíntese , RNA Catalítico/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR5/análise , Receptores CCR5/metabolismo , Receptores CXCR4/análise , Receptores CXCR4/metabolismo , Proteínas Virais/metabolismo , Replicação ViralRESUMO
The T-cell-specific retinoic acid receptor (RAR)-related orphan receptor-γ (RORγt) is a key transcription factor for the production of pro-inflammatory Th17 cytokines, which are implicated in the pathogenesis of autoimmune diseases. Over the years, several structurally diverse RORγt inverse agonists have been reported, but combining high potency and good physicochemical properties has remained a challenging task. We recently reported a new series of inverse agonists based on an imidazopyridine core with good physicochemical properties and excellent selectivity. Herein we report eight new X-ray crystal structures for different classes of natural and synthetic compounds, including examples selected from the patent literature. Analysis of their respective binding modes revealed insight into the molecular mechanisms that lead to agonism, antagonism, or inverse agonism. We report new molecular mechanisms for RORγt agonism and propose a separation of the inverse agonists into two classes: those that act via steric clash and those that act via other mechanisms (for the latter, co-crystallization with a co-activator peptide and helixâ 12 in the agonist position is still possible). For the non-steric clash inverse agonists, we propose a new mechanism ("water trapping") which can be combined with other mechanisms (e.g., close contacts with H479). In addition, we compare the interactions made for selected compounds in the "back pocket" near S404 and in the "sulfate pocket" near R364 and R367. Taken together, these new mechanistic insights should prove useful for the design and optimization of further RORγt modulators.
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Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/química , Proteínas Adaptadoras de Transdução de Sinal/química , Sítios de Ligação , Ésteres do Colesterol/química , Cristalografia por Raios X , Humanos , Hidrocarbonetos Fluorados/química , Imidazóis/química , Modelos Químicos , Proteínas Nucleares/química , Proteína 1 de Interação com Receptor Nuclear , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Piridinas/química , Sulfonamidas/química , Sulfonas/química , Água/químicaRESUMO
Retinoic acid receptor-related-orphan-receptor-C (RORγt) is the key transcription factor that is driving the differentiation of IL-17 producing T-helper 17 (Th17) cells that are implicated in the pathology of various autoimmune and inflammatory diseases. Based on the importance of RORγt in promoting Th17-driven pathology, there is considerable interest to develop low-molecular-weight compounds with the aim of inhibiting the transcriptional activity of this nuclear hormone receptor. In this article, we describe the in vitro and in vivo pharmacology of a potent and selective small-molecular-weight RORγt inverse agonist. The compound binds to the ligand binding domain (LBD) of RORγt leading to displacement of a co-activator peptide. We show for the first time that a RORγt inverse agonist down-regulates permissive histone H3 acetylation and methylation at the IL17A and IL23R promoter regions, thereby providing insight into the transcriptional inhibition of RORγt-dependent genes. Consistent with this, the compound effectively reduced IL-17A production by polarized human T-cells and γδT-cells and attenuated transcription of RORγt target genes. The inhibitor showed good in vivo efficacy in an antigen-induced arthritis model in rats and reduced the frequencies of IL-17A producing cells in ex vivo recall assays. In summary, we demonstrate that inhibiting RORγt by a low-molecular-weight inhibitor results in efficient and selective blockade of the pro-inflammatory Th17/IL-17A pathway making it an attractive target for Th17-mediated disorders.
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Artrite Experimental/tratamento farmacológico , Imidazóis/farmacologia , Interleucina-17/antagonistas & inibidores , Linfócitos Intraepiteliais/efeitos dos fármacos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Células Th17/efeitos dos fármacos , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Experimental/patologia , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imidazóis/síntese química , Interleucina-17/genética , Interleucina-17/imunologia , Linfócitos Intraepiteliais/imunologia , Linfócitos Intraepiteliais/patologia , Cinética , Masculino , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Regiões Promotoras Genéticas , Ligação Proteica , Piridinas/síntese química , Pirimidinas/síntese química , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Transdução de Sinais , Células Th17/imunologia , Células Th17/patologiaRESUMO
Retinoic-acid-orphan-receptor-C (RORC) is a master regulator of Th17 cells, which are pathogenic in several autoimmune diseases. Genetic Rorc deficiency in mice, while preventing autoimmunity, causes early lethality due to metastatic thymic T cell lymphomas. We sought to determine whether pharmacological RORC inhibition could be an effective and safe therapy for autoimmune diseases by evaluating its effects on Th17 cell functions and intrathymic T cell development. RORC inhibitors effectively inhibited Th17 differentiation and IL-17A production, and delayed-type hypersensitivity reactions. In vitro, RORC inhibitors induced apoptosis, as well as Bcl2l1 and BCL2L1 mRNA downregulation, in mouse and nonhuman primate thymocytes, respectively. Chronic, 13-week RORC inhibitor treatment in rats caused progressive thymic alterations in all analyzed rats similar to those in Rorc-deficient mice prior to T cell lymphoma development. One rat developed thymic cortical hyperplasia with preneoplastic features, including increased mitosis and reduced IKAROS expression, albeit without skewed T cell clonality. In summary, pharmacological inhibition of RORC not only blocks Th17 cell development and related cytokine production, but also recapitulates thymic aberrations seen in Rorc-deficient mice. While RORC inhibition may offer an effective therapeutic principle for Th17-mediated diseases, T cell lymphoma with chronic therapy remains an apparent risk.
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Receptores do Ácido Retinoico/antagonistas & inibidores , Células Th17/citologia , Timo/patologia , Animais , Regulação para Baixo , Feminino , Expressão Gênica , Humanos , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Receptores do Ácido Retinoico/genética , Células Th17/metabolismoRESUMO
Retinoic-acid-related orphan receptorâ γt (RORγt) is a key transcription factor implicated in the production of pro-inflammatory Th17 cytokines, which drive a number of autoimmune diseases. Despite diverse chemical series having been reported, combining high potency with a good physicochemical profile has been a very challenging task in the RORγt inhibitor field. Based on available chemical structures and incorporating in-house knowledge, a new series of triazolo- and imidazopyridine RORγt inverse agonists was designed. In addition, replacement of the terminal cyclopentylamide metabolic soft spot by five-membered heterocycles was investigated. From our efforts, we identified an optimal 6,7,8-substituted imidazo[1,2-a]pyridine core system and a 5-tert-butyl-1,2,4-oxadiazole as cyclopentylamide replacement leading to compounds 10 ((S)-N-(8-((4-(cyclopentanecarbonyl)-3-methylpiperazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide) and 33 ((S)-N-(8-((4-(5-(tert-butyl)-1,2,4-oxadiazol-3-yl)-3-methylpiperazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide). Both derivatives showed good pharmacological potencies in biochemical and cell-based assays combined with excellent physicochemical properties, including low to medium plasma protein binding across species. Finally, 10 and 33 were shown to be active in a rodent pharmacokinetic/pharmacodynamic (PK/PD) model after oral gavage at 15â mg kg-1 , lowering IL-17 cytokine production in exâ vivo antigen recall assays.
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Agonismo Inverso de Drogas , Imidazóis , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Piridinas/síntese química , Receptores do Ácido Retinoico/agonistas , Triazóis , Animais , Sítios de Ligação , Células Cultivadas , Cristalografia por Raios X , Humanos , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Concentração Inibidora 50 , Interleucina-17/sangue , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Piridinas/química , Piridinas/farmacologia , Ratos , Triazóis/síntese química , Triazóis/química , Triazóis/farmacologiaRESUMO
The balance between positive and negative signals plays a key role in determining T cell function. CTL-associated Ag-4 is a surface receptor that can inhibit T cell responses induced upon stimulation of the TCR and its CD28 coreceptor. Little is known regarding the signaling mechanisms elicited by CTLA-4. In this study we analyzed CTLA-4-mediated inhibition of TCR signaling in primary resting human CD4(+) T cells displaying low, but detectable, CTLA-4 cell surface expression. CTLA-4 coligation with the TCR resulted in reduced downstream protein tyrosine phosphorylation of signaling effectors and a striking inhibition of extracellular signal-regulated kinase 1/2 activation. Analysis of proximal TCR signaling revealed that TCR zeta-chain phosphorylation and subsequent zeta-associated protein of 70 kDa (ZAP-70) tyrosine kinase recruitment were not significantly affected by CTLA-4 engagement. However, the association of p56(lck) with ZAP-70 was inhibited following CTLA-4 ligation, correlating with reduced actions of p56(lck) in the ZAP-70 immunocomplex. Moreover, CTLA-4 ligation caused the selective inhibition of CD3-mediated phosphorylation of the positive regulatory ZAP-70 Y319 site. In addition, we demonstrate protein tyrosine phosphatase activity associated with the phosphorylated CTLA-4 cytoplasmic tail. The major phosphatase activity was attributed to Src homology protein 2 domain-containing tyrosine phosphatase 1, a protein tyrosine phosphatase that has been shown to be a negative regulator of multiple signaling pathways in hemopoietic cells. Collectively, our findings suggest that CTLA-4 can act early during the immune response to regulate the threshold of T cell activation.