RESUMO
Type 1 interferon (IFN) is a key mediator of organismal responses to pathogens, eliciting prototypical "interferon signature genes" that encode antiviral and inflammatory mediators. For a global view of IFN signatures and regulatory pathways, we performed gene expression and chromatin analyses of the IFN-induced response across a range of immunocyte lineages. These distinguished ISGs by cell-type specificity, kinetics, and sensitivity to tonic IFN and revealed underlying changes in chromatin configuration. We combined 1,398 human and mouse datasets to computationally infer ISG modules and their regulators, validated by genetic analysis in both species. Some ISGs are controlled by Stat1/2 and Irf9 and the ISRE DNA motif, but others appeared dependent on non-canonical factors. This regulatory framework helped to interpret JAK1 blockade pharmacology, different clusters being affected under tonic or IFN-stimulated conditions, and the IFN signatures previously associated with human diseases, revealing unrecognized subtleties in disease footprints, as affected by human ancestry.
Assuntos
Redes Reguladoras de Genes , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Conjuntos de Dados como Assunto , Humanos , Janus Quinases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Interferon alfa e beta/metabolismoRESUMO
In the last decade, mental toughness (MT) researchers have been interested in the behaviours exemplifying MT. Despite this interest, little attention has been paid to the competitive situations these behaviours occur in. Hence, the aim of the current study was to start addressing this gap by comprehensively focusing on the situations requiring MT in sport - specifically, the contextual demands linked to MT in women's Australian rules football. Focus groups and individual semi-structured interviews were conducted at two Australian rules football clubs after each competitive round of the 2020 season. Following analysis of participant responses, three broad situational categories were created, representing the psychological demands required by the player or team to successfully overcome in-game challenges. These categories were: context intelligence, attentional regulation, and emotional regulation. This study identified that situations requiring MT also required a degree of acceptance and commitment - alluding to a potential link between acceptance-commitment therapy (ACT) and MT. Conceptually, this view offers a new perspective on the psychological process of being mentally tough in competition. The link between ACT and MT also offers an avenue for MT development. Recommendations are made for incorporating these identified situations into regular training sessions following affective learning design principles.
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Desempenho Atlético , Esportes de Equipe , Feminino , Humanos , Atletas/psicologia , Desempenho Atlético/psicologia , AustráliaRESUMO
Herein, we disclose a new series of TYK2/ JAK1 inhibitors based upon a 3.1.0 azabicyclic substituted pyrimidine scaffold. We illustrate the use of structure-based drug design for the initial design and subsequent optimization of this series of compounds. One advanced example 19 met program objectives for potency, selectivity and ADME, and demonstrated oral activity in the adjuvant-induced arthritis rat model.
Assuntos
Artrite Experimental/tratamento farmacológico , Desenho de Fármacos , Janus Quinase 1/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , TYK2 Quinase/antagonistas & inibidores , Animais , Artrite Experimental/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Janus Quinase 1/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Ratos , Ratos Endogâmicos Lew , Relação Estrutura-Atividade , TYK2 Quinase/metabolismoRESUMO
Small-molecule inhibitors of the Janus kinase family (JAKis) are clinically efficacious in multiple autoimmune diseases, albeit with increased risk of certain infections. Their precise mechanism of action is unclear, with JAKs being signaling hubs for several cytokines. We assessed the in vivo impact of pan- and isoform-specific JAKi in mice by immunologic and genomic profiling. Effects were broad across the immunogenomic network, with overlap between inhibitors. Natural killer (NK) cell and macrophage homeostasis were most immediately perturbed, with network-level analysis revealing a rewiring of coregulated modules of NK cell transcripts. The repression of IFN signature genes after repeated JAKi treatment continued even after drug clearance, with persistent changes in chromatin accessibility and phospho-STAT responsiveness to IFN. Thus, clinical use and future development of JAKi might need to balance effects on immunological networks, rather than expect that JAKis affect a particular cytokine response and be cued to long-lasting epigenomic modifications rather than by short-term pharmacokinetics.
Assuntos
Citocinas/metabolismo , Inibidores de Janus Quinases/farmacologia , Janus Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Citocinas/genética , Redes Reguladoras de Genes/efeitos dos fármacos , Redes Reguladoras de Genes/imunologia , Fenômenos Imunogenéticos/efeitos dos fármacos , Fenômenos Imunogenéticos/genética , Janus Quinases/genética , Janus Quinases/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Transcriptoma/efeitos dos fármacos , Transcriptoma/imunologiaRESUMO
This study evaluated the short-term effects of tofacitinib treatment on peripheral blood leukocyte phenotype and function, and the reversibility of any such effects following treatment withdrawal in healthy volunteers. Cytomegalovirus (CMV)-seropositive subjects received oral tofacitinib 10â¯mg twice daily for 4â¯weeks and were followed for 4â¯weeks after drug withdrawal. There were slight increases in total lymphocyte and total T-cell counts during tofacitinib treatment, and B-cell counts increased by up to 26%. There were no significant changes in granulocyte or monocyte counts, or granulocyte function. Naïve and central memory T-cell counts increased during treatment, while all subsets of activated T cells were decreased by up to 69%. T-cell subsets other than effector memory cluster of differentiation (CD)4+, activated naïve CD4+ and effector CD8+ T-cell counts and B-cell counts, normalized 4â¯weeks after withdrawal. Following ex vivo activation, measures of CMV-specific T-cell responses, and antigen non-specific T-cell-mediated cytotoxicity and interferon (IFN)-γ production, decreased slightly. These T-cell functional changes were most pronounced at Day 15, partially normalized while still on tofacitinib and returned to baseline after drug withdrawal. Total natural killer (NK)-cell counts decreased by 33%, returning towards baseline after drug withdrawal. NK-cell function decreased during tofacitinib treatment, but without a consistent time course across measured parameters. However, markers of NK-cell-mediated cytotoxicity, antibody-dependent cellular cytotoxicity and IFN-γ production were decreased up to 42% 1â¯month after drug withdrawal. CMV DNA was not detectable in whole blood, and there were no cases of herpes zoster reactivation. No new safety concerns arose. In conclusion, the effect of short-term tofacitinib treatment on leukocyte composition and function in healthy CMV+ volunteers is modest and largely reversible 4â¯weeks after withdrawal.
Assuntos
Inibidores de Janus Quinases/farmacologia , Leucócitos/efeitos dos fármacos , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Adulto , Idoso , Artrite Reumatoide/tratamento farmacológico , Feminino , Voluntários Saudáveis , Humanos , Leucócitos/imunologia , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Fenótipo , Piperidinas/efeitos adversos , Pirimidinas/efeitos adversos , Pirróis/efeitos adversos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
BACKGROUND: Tofacitinib is an oral Janus kinase inhibitor being investigated for psoriasis. OBJECTIVE: We sought to elucidate the molecular mechanisms underlying the clinical efficacy of tofacitinib in patients with psoriasis. METHODS: Twelve patients with plaque psoriasis were randomized (3:1) to receive 10 mg of tofacitinib or placebo twice daily for 12 weeks. Biopsy specimens were taken from nonlesional (baseline) and lesional (baseline, days 1 and 3, and weeks 1, 2, 4, and 12) skin. Biopsy specimens were examined for psoriatic epidermal features (thickness, Ki67(+) keratinocytes and keratin 16 [KRT16] mRNA expression, and phosphorylated signal transducer and activator of transcription [pSTAT](+) nuclei) and T-cell and dendritic cell (DC) subsets by using immunohistochemistry, and mRNA transcripts were quantified by using a microarray. RESULTS: In lesional skin keratinocyte pSTAT1 and pSTAT3 staining was increased at baseline but reduced after 1 day of tofacitinib (baseline, median of 1290 pSTAT1(+) cells/µm(2); day 1, median of 332 pSTAT1(+) cells/µm(2); and nonlesional, median of 155 pSTAT1(+) cells/µm(2)). Epidermal thickness and KRT16 mRNA expression were significantly and progressively reduced after days 1 and 3 of tofacitinib administration, respectively (eg, KRT16 decreased 2.74-fold, day 3 vs baseline, P = .016). Decreases in DC and T-cell numbers were observed after weeks 1 and 2, respectively. At week 4, significant decreases in IL-23/TH17 pathways were observed that persisted through week 12. Improvements in clinical and histologic features were strongly associated with changes in expression of psoriasis-related genes and reduction in IL-17 gene expression. CONCLUSIONS: Tofacitinib has a multitiered response in patients with psoriasis: (1) rapid attenuation of keratinocyte Janus kinase/STAT signaling; (2) removal of keratinocyte-induced cytokine signaling, leading to reductions in pathologic DC and T-cell numbers to nonlesional levels; and (3) inhibition of the IL-23/TH17 pathway.
Assuntos
Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Psoríase/tratamento farmacológico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Biópsia , Método Duplo-Cego , Esquema de Medicação , Feminino , Humanos , Queratinócitos/imunologia , Queratinócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Psoríase/imunologia , Psoríase/metabolismo , Psoríase/patologia , Transdução de Sinais , Pele/imunologia , Pele/metabolismo , Pele/patologia , Resultado do Tratamento , Adulto JovemRESUMO
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterised by infiltration of immune cells into the affected synovium, release of inflammatory cytokines and degradative mediators, and subsequent joint damage. Both innate and adaptive arms of the immune response play a role, with activation of immune cells leading to dysregulated expression of inflammatory cytokines. Cytokines work within a complex regulatory network in RA, signalling through different intracellular kinase pathways to modulate recruitment, activation and function of immune cells and other leukocytes. As our understanding of RA has advanced, intracellular signalling pathways such as Janus kinase (JAK) pathways have emerged as key hubs in the cytokine network and, therefore, important as therapeutic targets. Tofacitinib is an oral JAK inhibitor for the treatment of RA. Tofacitinib is a targeted small molecule, and an innovative advance in RA therapy, which modulates cytokines critical to the progression of immune and inflammatory responses. Herein we describe the mechanism of action of tofacitinib and the impact of JAK inhibition on the immune and inflammatory responses in RA.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Janus Quinases/antagonistas & inibidores , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Artrite Reumatoide/imunologia , Citocinas/fisiologia , Humanos , Janus Quinases/fisiologia , Subpopulações de Linfócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Piperidinas/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Transdução de Sinais/fisiologiaRESUMO
Wild-caught crystal jellyfish (Aequorea victoria) arrived at the John G. Shedd Aquarium infested with hyperiid amphipods (Hyperia medusarum), which were inadvertently introduced into a system containing several jellyfish species. Affected systems were treated with milbemycin oxime (Interceptor tablets for dogs 51-100 lbs, Novartis Animal Health US, Inc., Greensboro, North Carolina 27408, USA), a treatment prescribed for red bug (Tegastes acroporanus) infestation in corals. Two treatments using one 25-mg aliquot of Interceptor per 10 gallons of tank water administered 6-7 days apart were completed. Overall, treatment to eradicate the parasite from the affected systems was successful. Further studies evaluating the tolerance of jellyfish to milbemycin oxime, particularly in small juvenile Eutonina indicans and Aurelia aurita, are warranted. Based on clinical observations, there were more negative effects associated with the treatment in the hydrozoans than in the scyphozoans.
Assuntos
Anfípodes/efeitos dos fármacos , Hidrozoários/parasitologia , Macrolídeos/uso terapêutico , Animais , Anti-Helmínticos/farmacologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Macrolídeos/administração & dosagemRESUMO
Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to PGs via the cyclooxygenase 1 and 2 pathways and to leukotrienes via the 5-lipoxygenase pathway. We used adoptive transfer and relapsing-remitting forms of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, in two different strains of mice (SJL or C57BL/6) to demonstrate that blockade of cPLA(2)α with a highly specific small-molecule inhibitor during the tissue-damage effector phase abrogates the clinical manifestation of disease. Using the adoptive transfer model in SJL mice, we demonstrated that the blockade of cPLA(2)α during the effector phase of disease was more efficacious in ameliorating the disease pathogenesis than the blockade of each of the downstream enzymes, cyclooxygenase-1/2 and 5-lipooxygenase. Similarly, blockade of cPLA(2)α was highly efficacious in ameliorating disease pathogenesis during the effector phase of EAE in the adoptive transfer model of EAE in C57BL/6 mice. Investigation of the mechanism of action indicates that cPLA(2)α inhibitors act on APCs to diminish their ability to induce Ag-specific effector T cell proliferation and proinflammatory cytokine production. Furthermore, cPLA(2)α inhibitors may prevent activation of CNS-resident microglia and may increase oligodendrocyte survival. Finally, in a relapsing-remitting model of EAE in SJL mice, therapeutic administration of a cPLA(2)α inhibitor, starting from the peak of disease or during remission, completely protected the mice from subsequent relapses.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Esclerose Múltipla/prevenção & controle , Transferência Adotiva , Animais , Células Apresentadoras de Antígenos/enzimologia , Células Apresentadoras de Antígenos/patologia , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/imunologia , Araquidonato 5-Lipoxigenase/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/imunologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2 , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Fosfolipases A2 do Grupo IV/genética , Fosfolipases A2 do Grupo IV/imunologia , Fosfolipases A2 do Grupo IV/metabolismo , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Microglia/enzimologia , Microglia/imunologia , Microglia/patologia , Esclerose Múltipla/enzimologia , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Oligodendroglia/enzimologia , Oligodendroglia/imunologia , Oligodendroglia/patologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
BACKGROUND: In recent years, the length of elite sporting competitions has raised concerns regarding player well-being, highlighting a need to review current match calendars. Therefore, this study aimed to explore the perceptions of elite National Rugby League (NRL) players and staff on the annual training and competition calendar from a player workload and well-being perspective. METHODS: This study adopted a mixed-methods approach, using a sequential explanatory design. Phase one implemented a cross-sectional survey, and phase two utilised semi-structured interviews. Four hundred and thirty-nine elite rugby league players and 46 staff completed the survey. Eighteen elite professional NRL players and six football staff were interviewed, and verbal data were analysed into pre-defined topic summaries using qualitative coding reliability methods. Topics included in-season, off-season, pre-season and well-being. RESULTS: Data analysis suggests that elite NRL players and staff believe players appear particularly comfortable with the current number of games; however, they are at their maximum capacity. Importantly, this study identified several minority groups that may require support to enhance player well-being. Players believe reducing the pre-season would negate fatigue experienced later in the subsequent season. Players and staff believe this timeframe still provides sufficient time to prepare for the upcoming season. Further, players were open to extending the off-season to 8-10 weeks and believed that extra time would allow for greater recovery from the previous season. Mid-season congested scheduling affects players following the intensified period and requires attention to alleviate fatigue. CONCLUSION: The results of this study convey important implications for the NRL, emphasising a need to review their annual training and competitive calendar, or to implement specific strategies to enhance the well-being of minority groups. The findings from this study should be considered when discussing the ideal length and structure of the match calendar to support players' physical and mental welfare.
RESUMO
Scholars have argued that mental toughness is a term that represents hypermasculine ideals. This hypermasculinity ideal could be considered relevant in the sport of Australian rules football, which has been played, at the professional level, by male athletes for the majority of its existence. Given the rising popularity of the Australian Football League Women's (AFLW), the present research sought to explore how the term mental toughness was understood at an AFLW club through a cultural sport psychology lens. Interviews were conducted with players (n = 4) and coaches (n = 6) from an AFLW club over the course of a competitive season. Data were transcribed verbatim and thematically analysed, with themes constructed to correspond with different levels of contextual influence. From this analysis, the club values and underlying assumptions, the social truths, and the role models/archetypes that provided a collective understanding of the term were identified. Mental toughness was defined by high-performance values of the men's game, which had implications for female athletes in this environment who are not afforded the same opportunities to fully embody these values. Mental toughness is positioned, in this environment, as an ideal with different meanings for female athletes due to structural factors associated with elite-level competition (e.g., full-time versus part-time professionalism).
Assuntos
Atletas , Homens , Humanos , Feminino , Masculino , Austrália , Esportes de EquipeRESUMO
Asthma is a chronic inflammatory lung disease with considerable unmet medical needs for new and effective therapies. Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme that is ultimately responsible for the production of eicosanoids implicated in the pathogenesis of asthma. We investigated a novel cPLA(2)α inhibitor, PF-5212372, to establish the potential of this drug as a treatment for asthma. PF-5212372 was a potent inhibitor of cPLA(2)α (7 nM) and was able to inhibit prostaglandin (PG)D(2) and cysteinyl leukotriene release from anti-IgE-stimulated human lung mast cells (0.29 and 0.45 nM, respectively). In a mixed human lung cell population, PF-5212372 was able to inhibit ionomycin-stimulated release of leukotriene B(4), thromboxane A(2), and PGD(2) (2.6, 2.6, and 4.0 nM, respectively) but was significantly less effective against PGE(2) release (>301 nM; p < 0.05). In an in vitro cell retention assay, PF-5212372 retained its potency up to 24 h after being washed off. In a sheep model of allergic inflammation, inhalation of PF-5212372 significantly inhibited late-phase bronchoconstriction (78% inhibition; p < 0.001) and airway hyper-responsiveness (94% inhibition; p < 0.001), and isolated sheep lung mast cell assays confirmed species translation via effective inhibition of PGD(2) release (0.78 nM). Finally, PF-5212372 was assessed for its ability to inhibit the contraction of human bronchi induced by AMP. PF5212372 significantly inhibited AMP-induced contraction of human bronchi (81% inhibition; p < 0.001); this finding, together with the ability of this drug to be effective in a wide range of preclinical asthma models, suggests that inhibition of cPLA(2)α with PF-5212372 may represent a new therapeutic option for the treatment of asthma.
Assuntos
Asma/tratamento farmacológico , Citosol/enzimologia , Inibidores Enzimáticos/uso terapêutico , Fosfolipases A2 do Grupo IV/antagonistas & inibidores , Fenilpropionatos/farmacologia , Sulfonamidas/farmacologia , Animais , Anticorpos Anti-Idiotípicos/farmacologia , Broncoconstrição/efeitos dos fármacos , Ionóforos de Cálcio/farmacologia , Linhagem Celular , Humanos , Mastócitos/fisiologia , Prostaglandina D2/metabolismo , OvinosRESUMO
Experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the central nervous system (CNS), is a model of human multiple sclerosis. Cytosolic phospholipase A2alpha (cPLA2alpha), which initiates production of prostaglandins, leukotrienes, and platelet-activating factor, is present in EAE lesions. Using myelin oligodendrocyte glycoprotein (MOG) immunization, as well as an adoptive transfer model, we showed that cPLA2alpha-/- mice are resistant to EAE. Histologic examination of the CNS from MOG-immunized mice revealed extensive inflammatory lesions in the cPLA2alpha+/- mice, whereas the lesions in cPLA2alpha-/- mice were reduced greatly or completely absent. MOG-specific T cells generated from WT mice induced less severe EAE in cPLA2alpha-/- mice compared with cPLA2alpha+/- mice, which indicates that cPLA2alpha plays a role in the effector phase of EAE. Additionally, MOG-specific T cells from cPLA2alpha-/- mice, transferred into WT mice, induced EAE with delayed onset and lower severity compared with EAE that was induced by control cells; this indicates that cPLA2alpha also plays a role in the induction phase of EAE. MOG-specific T cells from cPLA2alpha-/- mice were deficient in production of Th1-type cytokines. Consistent with this deficiency, in vivo administration of IL-12 rendered cPLA2alpha-/- mice susceptible to EAE. Our data indicate that cPLA2alpha plays an important role in EAE development and facilitates differentiation of T cells toward the Th1 phenotype.
Assuntos
Diferenciação Celular/imunologia , Citosol/enzimologia , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/genética , Fosfolipases A/deficiência , Células Th1/imunologia , Animais , Encefalomielite Autoimune Experimental/imunologia , Feminino , Fosfolipases A2 do Grupo IV , Imunidade Inata/genética , Imunofenotipagem , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas da Mielina , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Fosfolipases A/genética , Fosfolipases A/metabolismo , Fosfolipases A2 , Medula Espinal/imunologia , Medula Espinal/patologia , Células Th1/citologiaRESUMO
OBJECTIVE: All gamma-chain cytokines signal through JAK-3 and JAK-1 acting in tandem. We undertook this study to determine whether the JAK-3 selective inhibitor WYE-151650 would be sufficient to disrupt cytokine signaling and to ameliorate autoimmune disease pathology without inhibiting other pathways mediated by JAK-1, JAK-2, and Tyk-2. METHODS: JAK-3 kinase selective compounds were characterized by kinase assay and JAK-3-dependent (interleukin-2 [IL-2]) and -independent (IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF]) cell-based assays measuring proliferation or STAT phosphorylation. In vivo, off-target signaling was measured by IL-22- and erythropoietin (EPO)-mediated models, while on-target signaling was measured by IL-2-mediated signaling. Efficacy of JAK-3 inhibitors was determined using delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA) models in mice. RESULTS: In vitro, WYE-151650 potently suppressed IL-2-induced STAT-5 phosphorylation and cell proliferation, while exhibiting 10-29-fold less activity against JAK-3-independent IL-6- or GM-CSF-induced STAT phosphorylation. Ex vivo, WYE-151650 suppressed IL-2-induced STAT phosphorylation, but not IL-6-induced STAT phosphorylation, as measured in whole blood. In vivo, WYE-151650 inhibited JAK-3-mediated IL-2-induced interferon-gamma production and decreased the natural killer cell population in mice, while not affecting IL-22-induced serum amyloid A production or EPO-induced reticulocytosis. WYE-151650 was efficacious in mouse DTH and CIA models. CONCLUSION: In vitro, ex vivo, and in vivo assays demonstrate that WYE-151650 is efficacious in mouse CIA despite JAK-3 selectivity. These data question the need to broadly inhibit JAK-1-, JAK-2-, or Tyk-2-dependent cytokine pathways for efficacy.
Assuntos
Artrite Experimental/tratamento farmacológico , Janus Quinase 3/antagonistas & inibidores , Análise de Variância , Animais , Artrite Experimental/metabolismo , Western Blotting , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Janus Quinase 3/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis (RA), psoriatic arthritis, and ulcerative colitis, and has been previously investigated for psoriasis (PsO). This meta-analysis of genome-wide association studies (GWAS) was performed to identify genetic factors associated with increased risk/faster onset of herpes zoster (HZ) in subjects with RA or PsO receiving tofacitinib treatment, and to determine potential mechanisms that could be attributed to the varying rates of HZ across ethnicities. METHODS: In an ethnicity/indication-specific, trans-ethnic, trans-population meta-analysis of GWAS in subjects with RA or PsO from phase II, phase III, and long-term extension studies of tofacitinib, 8 million genetic variants were evaluated for their potential association with time to an HZ event and incidence of an HZ event (case versus control) with tofacitinib treatment, using Cox proportional hazard and logistic regression analyses, respectively. RESULTS: In total, 5,246 subjects were included (3,168 with RA and 2,078 with PsO). After adjustment for age, baseline absolute lymphocyte count, genetically defined ethnicity, and concomitant methotrexate use (in RA subjects only), 4 loci were significantly associated with faster onset of HZ in European subjects (P < 5 × 10-8 ), including a single-nucleotide polymorphism (SNP) near CD83 (frequency of risk allele ~2% in European subjects versus ~0.1% in East Asian subjects). In the trans-ethnic, trans-population meta-analysis, the CD83 SNP remained significant. Four additional significant loci were identified in the meta-analysis, among which a SNP near IL17RB was associated with faster onset of HZ (meta-analysis hazard ratio 3.6 [95% confidence interval 2.40-5.44], P = 7.6 × 10-10 ; frequency of risk allele ~12% in East Asian subjects versus <0.2% in European subjects). CONCLUSION: Genetic analysis of tofacitinib-treated subjects with RA or PsO identified multiple loci associated with increased HZ risk. Prevalent variants near the immune-relevant genes CD83 and IL17RB in European and East Asian populations, respectively, may contribute to risk of HZ in tofacitinib-treated subjects.
Assuntos
Antígenos CD/genética , Artrite Reumatoide/tratamento farmacológico , Herpes Zoster/genética , Imunoglobulinas/genética , Inibidores de Janus Quinases/efeitos adversos , Glicoproteínas de Membrana/genética , Piperidinas/efeitos adversos , Psoríase/tratamento farmacológico , Pirimidinas/efeitos adversos , Receptores de Interleucina-17/genética , Povo Asiático/genética , Estudo de Associação Genômica Ampla , Herpes Zoster/induzido quimicamente , Herpes Zoster/epidemiologia , Humanos , Modelos Logísticos , Polimorfismo de Nucleotídeo Único , Modelos de Riscos Proporcionais , População Branca/genética , Antígeno CD83RESUMO
Pathogenic mechanisms relevant to rheumatoid arthritis occur in the mouse model of collagen-induced arthritis (CIA). Cytosolic phospholipase A2alpha (cPLA2alpha) releases arachidonic acid from cell membranes to initiate the production of prostaglandins and leukotrienes. These inflammatory mediators have been implicated in the development of CIA. To test the hypothesis that cPLA2alpha plays a key role in the development of CIA, we backcrossed cPLA2alpha-deficient mice on the DBA/1LacJ background that is susceptible to CIA. The disease severity scores and the incidence of disease were markedly reduced in cPLA2alpha-deficient mice compared with wild-type littermates. At completion of the study, >90% of the wild-type mice had developed disease whereas none of the cPLA2alpha-deficient mice had more than one digit inflamed. Furthermore, visual disease scores correlated with severity of disease determined histologically. Pannus formation, articular fibrillation, and ankylosis were all dramatically reduced in the cPLA2alpha-deficient mice. Although the disease scores differed significantly between cPLA2alpha mutant and wild-type mice, anti-collagen antibody levels were similar in the wild-type mice and mutant littermates. These data demonstrate the critical role of cPLA2alpha in the pathogenesis of CIA.
Assuntos
Artrite/etiologia , Colágeno/imunologia , Citosol/enzimologia , Fosfolipases A/fisiologia , Proteínas Ativadoras de 5-Lipoxigenase , Animais , Artrite/prevenção & controle , Proteínas de Transporte/fisiologia , Ciclo-Oxigenase 2 , Fosfolipases A2 do Grupo IV , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Isoenzimas/fisiologia , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fosfolipases A/deficiência , Prostaglandina-Endoperóxido Sintases/fisiologia , Receptores de Prostaglandina E/fisiologia , Receptores de Prostaglandina E Subtipo EP4RESUMO
The IL-23/T helper type 17 cell axis is a target for psoriasis. The TYK2/Janus kinase 1 inhibitor PF-06700841 will directly suppress TYK2-dependent IL-12 and IL-23 signaling and Janus kinase 1-dependent signaling in cells expressing these signaling molecules, including T cells and keratinocytes. This clinical study sought to define the inflammatory gene and cellular pathways through which PF-06700841 improves the clinical manifestations of psoriasis. Patients (n = 30) with moderate-to-severe psoriasis were randomized to once-daily 30 mg (n = 14) or 100 mg (n = 7) PF-06700841 or placebo (n = 9) for 28 days. Biopsies were taken from nonlesional and lesional skin at baseline and weeks 2 and 4. Changes in the psoriasis transcriptome and genes induced by IL-17 in keratinocytes were evaluated with microarray profiling and reverse transcriptase-PCR. Reductions in IL-17A, IL-17F, and IL-12B mRNA were observed as early as 2 weeks and approximately 70% normalization of lesional gene expression after 4 weeks. Immunohistochemistry showed significant decreases in markers of keratinocyte activation, epidermal thickness, KRT16 and Ki-67 expression, and immune cell infiltrates CD3+/CD8+ (T cells) and CD11c (dendritic cells) after 2 weeks of treatment, corresponding with improvement in histologic score. PF-06700841 improves clinical symptoms of chronic plaque psoriasis by inhibition of proinflammatory cytokines that require TYK2 and Janus kinase 1 for signal transduction.
Assuntos
Inibidores de Proteínas Quinases/administração & dosagem , Psoríase/tratamento farmacológico , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Células Th17/efeitos dos fármacos , Adulto , Biópsia , Feminino , Seguimentos , Perfilação da Expressão Gênica , Humanos , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-17/metabolismo , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/metabolismo , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/efeitos adversos , Psoríase/imunologia , Psoríase/patologia , Pirazóis/efeitos adversos , Pirimidinas/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , TYK2 Quinase/antagonistas & inibidores , TYK2 Quinase/metabolismo , Células Th17/imunologia , Resultado do Tratamento , Adulto JovemRESUMO
Translation of modulation of drug target activity to therapeutic effect is a critical aspect for all drug discovery programs. In this work we describe the profiling of a non-receptor tyrosine-protein kinase (TYK2) inhibitor which shows a functionally relevant potency shift between human and preclinical species (e.g. murine, dog, macaque) in both biochemical and cellular assays. Comparison of the structure and sequence homology of TYK2 between human and preclinical species within the ATP binding site highlights a single amino acid (I960 â V) responsible for the potency shift. Through TYK2 kinase domain mutants and a TYK2 980I knock-in mouse model, we demonstrate that this single amino acid change drives a functionally relevant potency difference that exists between human and all evaluated preclinical species, for a series of TYK2 inhibitors which target the ATP binding site.
Assuntos
Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , TYK2 Quinase/antagonistas & inibidores , TYK2 Quinase/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/efeitos dos fármacos , Cães , Humanos , Janus Quinase 1 , Macaca , Camundongos , Mutação , Domínios Proteicos/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , TYK2 Quinase/genética , TYK2 Quinase/metabolismoRESUMO
Tyrosine kinase 2 (TYK2) is a member of the JAK kinase family that regulates signal transduction downstream of receptors for the IL-23/IL-12 pathways and type I interferon family, where it pairs with JAK2 or JAK1, respectively. On the basis of human genetic and emerging clinical data, a selective TYK2 inhibitor provides an opportunity to treat autoimmune diseases delivering a potentially differentiated clinical profile compared to currently approved JAK inhibitors. The discovery of an ATP-competitive pyrazolopyrazinyl series of TYK2 inhibitors was accomplished through computational and structurally enabled design starting from a known kinase hinge binding motif. With understanding of PK/PD relationships, a target profile balancing TYK2 potency and selectivity over off-target JAK2 was established. Lead optimization involved modulating potency, selectivity, and ADME properties which led to the identification of the clinical candidate PF-06826647 (22).
Assuntos
Doenças Autoimunes/enzimologia , Descoberta de Drogas/métodos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , TYK2 Quinase/antagonistas & inibidores , Animais , Doenças Autoimunes/tratamento farmacológico , Humanos , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Inibidores de Proteínas Quinases/uso terapêutico , Estrutura Secundária de Proteína , TYK2 Quinase/química , TYK2 Quinase/metabolismoRESUMO
The synthesis and optimization of a class of trisubstituted quinazoline-2,4(1H,3H)-dione cPLA(2)alpha inhibitors are described. Utilizing pharmacophores that were found to be important in our indole series, we discovered inhibitors with reduced lipophilicity and improved aqueous solubility. These compounds are active in whole blood assays, and cell-based assay results indicate that prevention of arachidonic acid release arises from selective cPLA(2)alpha inhibition.