RESUMO
Mouse and human data implicate the NOD1 and NOD2 sensors of the intestinal microbiome and the associated signal transduction via the receptor interacting protein kinase 2 (RIPK2) as a potential key signaling node for the development of inflammatory bowel disease (IBD) and an attractive target for pharmacological intervention. The TRUC mouse model of IBD was strongly indicated for evaluating RIPK2 antagonism for its effect on intestinal inflammation based on previous knockout studies with NOD1, NOD2, and RIPK2. We identified and profiled the BI 706039 molecule as a potent and specific functional inhibitor of both human and mouse RIPK2 and with favorable pharmacokinetic properties. We dosed BI 706039 in the spontaneous TRUC mouse model from age 28 to 56 days. Oral, daily administration of BI 706039 caused dose-responsive and significant improvement in colonic histopathological inflammation, colon weight, and terminal levels of protein-normalized fecal lipocalin (all P values <0.001). These observations correlated with dose responsively increasing systemic levels of the BI 706039 compound, splenic molecular target engagement of RIPK2, and modulation of inflammatory genes in the colon. This demonstrates that a relatively low oral dose of a potent and selective RIPK2 inhibitor can modulate signaling in the intestinal immune system and significantly improve disease associated intestinal inflammation.NEW & NOTEWORTHY The RIPK2 kinase at the apex of microbiome immunosensing is an attractive target for pharmacological intervention. A low oral dose of a RIPK2 inhibitor leads to significantly improved intestinal inflammation in the murine TRUC model of colitis. A selective and potent inhibitor of the RIPK2 kinase may represent a new class of therapeutics that target microbiome-driven signaling for the treatment of IBD.
Assuntos
Colite Ulcerativa/tratamento farmacológico , Colo/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Animais , Disponibilidade Biológica , Células Cultivadas , Colite Ulcerativa/enzimologia , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Colo/enzimologia , Colo/patologia , Doença de Crohn/enzimologia , Doença de Crohn/patologia , Citocinas/genética , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Fezes/química , Humanos , Mediadores da Inflamação/metabolismo , Lipocalinas/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Biológicos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proteínas com Domínio T/genéticaRESUMO
GPBAR1 is a G protein-coupled receptor that is activated by certain bile acids and plays an important role in the regulation of bile acid synthesis, lipid metabolism, and energy homeostasis. Recent evidence suggests that GPBAR1 may also have important effects in reducing the inflammatory response through its expression on monocytes and macrophages. To further understand the role of GPBAR1 in inflammation, we generated a novel, selective, proprietary GPBAR1 agonist and tested its effectiveness at reducing monocyte and macrophage activation in vitro and in vivo. We have used this agonist, together with previously described agonists to study agonism of GPBAR1, and shown that they can all induce cAMP and reduce TLR activation-induced cytokine production in human monocytes and monocyte-derived macrophages in vitro. Additionally, through the usage of RNA sequencing (RNA-Seq), we identified a select set of genes that are regulated by GPBAR1 agonism during LPS activation. To further define the in vivo role of GPBAR1 in inflammation, we assessed GPBAR1 expression and found high levels on circulating mouse monocytes. Agonism of GPBAR1 reduced LPS-induced cytokine production in mouse monocytes ex vivo and serum cytokine levels in vivo. Agonism of GPBAR1 also had profound effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, where monocytes play an important role. Mice treated with the GPBAR1 agonist exhibited a significant reduction in the EAE clinical score which correlated with reduced monocyte and microglial activation and reduced trafficking of monocytes and T cells into the CNS. These data confirm the importance of GPBAR1 in controlling monocyte and macrophage activation in vivo and support the rationale for selective agonists of GPBAR1 in the treatment of inflammatory diseases.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Receptores Acoplados a Proteínas G/agonistas , Animais , Células CHO , Análise por Conglomerados , Cricetulus , AMP Cíclico , Citocinas/biossíntese , Encefalomielite Autoimune Experimental/tratamento farmacológico , Feminino , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Células Mieloides/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Sphingosine-1-phosphate (S1P) is a bioactive metabolite with pleiotropic effects on multiple cellular processes in health and disease. Responses elicited by S1P are a result of binding to five specific G-protein-coupled receptors. We have developed multiple assays to systematically study the downstream signaling of these receptors, including early events such as direct receptor activation (GTPγS) as well as more distal events such as S1P1 receptor degradation. Employing such assays, we have characterized and compared multiple S1P1 agonists that are in clinical development including FTY720, BAF312, CS-0777, and other molecules from the S1P1 patent literature. Our parallel assessment has allowed us to compare their potency against S1P1, their selectivity against the four other S1P receptors, as well as species cross-reactivity. We note that all of the compounds studied signal in an identical manner through S1P1, leading to receptor degradation.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Animais , Células CHO , Cálcio/metabolismo , Linhagem Celular , Cricetulus , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas , Humanos , Camundongos , Organofosfatos/farmacologia , Proteólise/efeitos dos fármacos , Ratos , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologiaRESUMO
Sphingosine-1-phosphate (S1P) receptors are critical for lymphocyte egress from secondary lymphoid organs, and S1P receptor modulators suppress lymphocyte circulation. However, the role of S1P receptors on monocytes is less clear. To elucidate this, we systematically evaluated monocytes in rats and mice, both in naive and inflammatory conditions, with S1P receptor modulators FTY720 and BAF312. We demonstrate that S1P receptor modulators reduce circulating monocytes in a similar time course as lymphocytes. Furthermore, total monocyte numbers were increased in the spleen and bone marrow, suggesting that S1P receptor modulation restricts egress from hematopoietic organs. Monocytes treated ex vivo with FTY720 had reduced CD40 expression and TNF-α production, suggesting a direct effect on monocyte activation. Similar reductions in protein expression and cytokine production were also found in vivo. Suppression of experimental autoimmune encephalomyelitis in mice and rats by FTY720 correlated with reduced numbers of lymphocytes and monocytes. These effects on monocytes were independent of S1P3, as treatment with BAF312, a S1P1,4,5 modulator, led to similar results. These data reveal a novel role for S1P receptors on monocytes and offer additional insights on the mechanism of action of S1P receptor modulators in disease.
Assuntos
Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Propilenoglicóis/farmacologia , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Movimento Celular/imunologia , Citocinas/biossíntese , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Cloridrato de Fingolimode , Células Matadoras Naturais/metabolismo , Contagem de Leucócitos , Camundongos , Monócitos/imunologia , Neutrófilos/metabolismo , Ratos , Esfingosina/farmacologia , Baço/efeitos dos fármacos , Baço/metabolismoRESUMO
Cardiovascular disease (CVD) biomarkers were examined in a cohort of HIV-infected and HIV-uninfected adolescents who participated in Adolescent Trials Network study 083 utilizing samples from the Reaching for Excellence in Adolescent Care cohort, a longitudinal study of youth infected through adult risk behavior. Nonfasting blood samples from 97 HIV-infected and 81 HIV-uninfected adolescents infected by adult risk behaviors were analyzed for total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), very low-density lipoprotein (VLDL), triglycerides, apolipoprotein A-I, high-sensitivity C-reactive protein (hsCRP), soluble vascular adhesion molecule-1 (sVCAM-1), myeloperoxidase, and neopterin at baseline and 18 months later. Results were analyzed using ANOVA, Wilcoxon signed-rank, and paired t tests. Among infected subjects 67 received antiretroviral therapy and 30 were treatment naive. The HIV-infected and HIV-uninfected subjects were similar in gender, ethnicity, and cardiovascular risk factors such as smoking and obesity. In all groups lipid parameters were within accepted guidelines for cardiovascular risk. Among HIV-infected youth on antiretroviral therapy (ART), HDL and apoprotein A-I were significantly lower when compared to uninfected youth. hsCRP was not elevated and thus not predictive for risk in any group. sVCAM-1 levels were significantly elevated in both HIV-infected groups: 1,435 ng/ml and 1,492 ng/ml in untreated and treated subjects, respectively, and 1,064 ng/ml in the uninfected group (p<0.0001). Across all groups neopterin correlated with sVCAM at 18 months (Spearman correlation coefficient 0.58, p<0.0001). Only 9% of ART-treated subjects fully suppressed virus. Lipid profiles and hsCRP, traditional markers of cardiovascular disease, are not abnormal among HIV-infected youth but elevated sVCAM may be an early marker of atherosclerosis.
Assuntos
Biomarcadores/sangue , Doenças Cardiovasculares/diagnóstico , Endotélio Vascular/patologia , Infecções por HIV/complicações , Infecções por HIV/virologia , HIV-1/patogenicidade , Molécula 1 de Adesão de Célula Vascular/sangue , Adolescente , Estudos de Coortes , Feminino , Humanos , Estudos Longitudinais , MasculinoRESUMO
A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.
Assuntos
Compostos de Bifenilo/metabolismo , Inibidores Enzimáticos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Estrutura Terciária de Proteína , Fator 2 Ativador da Transcrição , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Compostos de Bifenilo/química , Calorimetria , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Inibidores Enzimáticos/química , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteína Quinase 14 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Fosforilação , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Especificidade por Substrato , Ressonância de Plasmônio de Superfície , Fatores de Transcrição/metabolismoRESUMO
The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.
Assuntos
Benzimidazóis/síntese química , Inibidores Enzimáticos/síntese química , Imunossupressores/síntese química , Isoquinolinas/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Administração Oral , Animais , Anticorpos Monoclonais/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Complexo CD3/imunologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imunossupressores/química , Imunossupressores/farmacologia , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Interleucina-2/sangue , Isoquinolinas/química , Isoquinolinas/farmacologia , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
An imidazo[4,5-h]isoquinolin-7,9-dione (1) was identified as an adenosine 5'-triphosphate competitive inhibitor of lck by high throughput screening. Initial structure-activity relationship studies identified the dichlorophenyl ring and the imide NH as important pharmacophores. A binding model was constructed to understand how 1 binds to a related kinase, hck. These results suggested that removing the gem-dimethyl group and flattening the ring would enhance activity. This was realized by converting 1 to the imidazo[4,5-h]isoquinolin-9-one (20), resulting in an 18-fold improvement in potency against lck and a 50-fold increase in potency in a cellular assay.