RESUMO
Optimization of the potency and pharmacokinetic profile of 2,3,4-trisubstituted quinoline, 4, led to the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 6a (AM-0687) and 7 (AM-1430). On the basis of their improved profile, these analogs were selected for in vivo pharmacodynamic (PD) and efficacy experiments in animal models of inflammation. The in vivo PD studies, which were carried out in a mouse pAKT inhibition animal model, confirmed the observed potency of 6a and 7 in biochemical and cellular assays. Efficacy experiments in a keyhole limpet hemocyanin model in rats demonstrated that administration of either 6a or 7 resulted in a strong dose-dependent reduction of IgG and IgM specific antibodies. The excellent in vitro and in vivo profiles of these analogs make them suitable for further development.
Assuntos
Descoberta de Drogas , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Quinolinas/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Relação Dose-Resposta a Droga , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Piridinas/síntese química , Piridinas/química , Quinolinas/síntese química , Quinolinas/química , Relação Estrutura-AtividadeRESUMO
Lead optimization efforts resulted in the discovery of two potent, selective, and orally bioavailable PI3Kδ inhibitors, 1 (AM-8508) and 2 (AM-9635), with good pharmacokinetic properties. The compounds inhibit B cell receptor (BCR)-mediated AKT phosphorylation (pAKT) in PI3Kδ-dependent in vitro cell based assays. These compounds which share a benzimidazole bicycle are effective when administered in vivo at unbound concentrations consistent with their in vitro cell potency as a consequence of improved unbound drug concentration with lower unbound clearance. Furthermore, the compounds demonstrated efficacy in a Keyhole Limpet Hemocyanin (KLH) study in rats, where the blockade of PI3Kδ activity by inhibitors 1 and 2 led to effective inhibition of antigen-specific IgG and IgM formation after immunization with KLH.
Assuntos
Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Animais , Linfócitos B/efeitos dos fármacos , Cristalografia por Raios X , Hemocianinas/efeitos dos fármacos , Humanos , Imunoglobulina G/efeitos dos fármacos , Imunoglobulina M/efeitos dos fármacos , Camundongos , Modelos Moleculares , Ratos , Relação Estrutura-AtividadeRESUMO
Asthma is a heterogeneous disease characterized by airway inflammation and hyperreactivity. IL-17 receptor A (IL-17RA) is a shared receptor subunit required for activity of IL-17 family cytokines, including IL-17A and IL-25. IL-17A and IL-25 induce different proinflammatory responses, and concentrations are elevated in subjects with asthma. However, the individual contributions of IL-17A and IL-25 to disease pathogenesis are unclear. We explored proinflammatory activities of the IL-17 pathway in models of pulmonary inflammation and assessed its effects on contractility of human bronchial airway smooth muscle. In two mouse models, IL-17RA, IL-17RB, or IL-25 blockade reduced airway inflammation and airway hyperreactivity. Individually, IL-17A and IL-25 enhanced contractility of human bronchial smooth muscle induced by methacholine or carbachol. IL-17A had more pronounced effects on methacholine-induced contractility in bronchial rings from donors with asthma compared with donors without asthma. Blocking the IL-17 pathway via IL-17RA may be a useful therapy for some patients with asthma by reducing pulmonary inflammation and airway hyperreactivity.
Assuntos
Asma/metabolismo , Receptores de Interleucina-17/fisiologia , Animais , Asma/imunologia , Brônquios/imunologia , Brônquios/patologia , Células Cultivadas , Expressão Gênica , Humanos , Interleucina-17/fisiologia , Interleucinas/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismo , Transdução de SinaisRESUMO
2,3,4-Substituted quinolines such as (10a) were found to be potent inhibitors of PI3Kδ in both biochemical and cellular assays with good selectivity over three other class I PI3K isoforms. Some of those analogs showed favorable pharmacokinetic properties.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Animais , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Masculino , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Quinolinas/síntese química , Quinolinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
The development and optimization of a series of quinolinylpurines as potent and selective PI3Kδ kinase inhibitors with excellent physicochemical properties are described. This medicinal chemistry effort led to the identification of 1 (AMG319), a compound with an IC50 of 16 nM in a human whole blood assay (HWB), excellent selectivity over a large panel of protein kinases, and a high level of in vivo efficacy as measured by two rodent disease models of inflammation.
Assuntos
Adenosina/farmacologia , Doenças Autoimunes/prevenção & controle , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inflamação/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Quinolinas/farmacologia , Adenosina/química , Adenosina/metabolismo , Animais , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases/química , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Cristalografia por Raios X , Modelos Animais de Doenças , Descoberta de Drogas , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Quinolinas/química , Quinolinas/metabolismo , Ratos Endogâmicos Lew , Células Sf9 , Relação Estrutura-AtividadeRESUMO
Structure-based rational design led to the synthesis of a novel series of potent PI3K inhibitors. The optimized pyrrolopyridine analogue 63 was a potent and selective PI3Kß/δ dual inhibitor that displayed suitable physicochemical properties and pharmacokinetic profile for animal studies. Analogue 63 was found to be efficacious in animal models of inflammation including a keyhole limpet hemocyanin (KLH) study and a collagen-induced arthritis (CIA) disease model of rheumatoid arthritis. These studies highlight the potential therapeutic value of inhibiting both the PI3Kß and δ isoforms in the treatment of a number of inflammatory diseases.
Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Modelos MolecularesAssuntos
Doenças Autoimunes/enzimologia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Inflamação/enzimologia , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Doenças Autoimunes/tratamento farmacológico , Linfócitos B/enzimologia , Classe Ia de Fosfatidilinositol 3-Quinase/química , Classe Ib de Fosfatidilinositol 3-Quinase/química , Humanos , Inflamação/tratamento farmacológico , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Terapia de Alvo Molecular , Neutrófilos/enzimologia , Conformação Proteica , Linfócitos T/enzimologiaRESUMO
The ICOS (Inducible T cell Co-Stimulator)/B7RP-1 (B7-related protein 1) interaction is critical for the proper activation of a T lymphocyte. In this manuscript we describe a systematic in vivo approach to determine the level of blockade required to impair the generation of a T cell-dependent antibody response. We have developed an overall strategy for correlating drug exposure, target saturation, and efficacy in a biological response that can be generalized for most protein therapeutics. Using this strategy, we determined that low levels of B7RP-1 blockade are still sufficient to inhibit the immune response. These data suggest that contact between the T cell and the antigen-presenting cell during antigen presentation is much more sensitive to inhibition than previously believed and that ICOS/B7RP-1 blockade may be efficacious in the treatment of autoimmune diseases.
Assuntos
Antígeno B7-1/farmacologia , Fenômenos do Sistema Imunitário/efeitos dos fármacos , Hidróxido de Alumínio/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Células Apresentadoras de Antígenos/imunologia , Antígenos CD19/metabolismo , Linfócitos B/metabolismo , Antígeno B7-1/genética , Sítios de Ligação , Complexo CD3/metabolismo , Citocinas/sangue , Relação Dose-Resposta a Droga , Feminino , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/metabolismo , Hemocianinas/imunologia , Ligante Coestimulador de Linfócitos T Induzíveis , Camundongos , Camundongos Endogâmicos BALB C , Modelos Imunológicos , Ligação Proteica , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/metabolismo , Temperatura , Fatores de TempoRESUMO
Autoimmune diseases are marked by the presence of class-switched, high-affinity autoantibodies with pathogenic potential. Costimulation plays an important role in the activation of T cells and the development of T cell-dependent B cell responses. ICOS plays an indispensable role in the development of follicular helper T cells (T(FH) cells), which provide cognate help to germinal center (GC) B cells. We show that the levels of T(FH) cells and GC B cells in two different models of autoimmunity, the New Zealand Black/New Zealand White (NZB/NZW) F(1) mouse model of systemic lupus erythematosus and the collagen-induced arthritis model of rheumatoid arthritis, are dependent on the maintenance of the ICOS/B7RP-1 pathway. Treatment with an anti-B7RP-1 Ab ameliorates disease manifestations and leads to a decrease in T(FH) cells and GC B cells as well as an overall decrease in the frequency of ICOS(+) T cells. Coculture experiments of Ag-primed B cells with CXCR5(+) or CXCR5(-) T cells show that blocking B7RP-1 does not directly impact the production of IgG by B cells. These findings further support the role of ICOS in autoimmunity and suggest that the expansion of the T(FH) cell pool is an important mechanism by which ICOS regulates Ab production.
Assuntos
Anticorpos Bloqueadores/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Autoanticorpos/biossíntese , Antígeno B7-1/imunologia , Diferenciação Celular/imunologia , Centro Germinativo/imunologia , Transdução de Sinais/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/fisiologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Experimental/terapia , Autoanticorpos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Antígeno B7-1/fisiologia , Feminino , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Ligante Coestimulador de Linfócitos T Induzíveis , Proteína Coestimuladora de Linfócitos T Induzíveis , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Lúpus Eritematoso Sistêmico/terapia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Endogâmicos NZB , Distribuição Aleatória , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
The lymphocyte-specific kinase (Lck), a member of the Src family of cytoplasmic tyrosine kinases, is expressed in T cells and natural killer (NK) cells. Genetic evidence, including knockout mice and human mutations, demonstrates that Lck kinase activity is critical for normal T cell development, activation, and signaling. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. With the aid of X-ray structure-based analysis, aminopyrimidine amides 2 and 3 were designed from aminoquinazolines 1, which had previously been demonstrated to exhibit potent inhibition of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminopyrimidine amides 3 possessing improved cellular potency and selectivity profiles relative to their aminoquinazoline predecessors 1. Orally bioavailable compound 13b inhibited the anti-CD3-induced production of interleukin-2 (IL-2) in mice in a dose-dependent manner (ED 50 = 9.4 mg/kg).
Assuntos
Amidas/farmacologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Linfócitos T/efeitos dos fármacos , Administração Oral , Amidas/síntese química , Amidas/química , Animais , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Interleucina-2/antagonistas & inibidores , Interleucina-2/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Estereoisomerismo , Relação Estrutura-Atividade , Linfócitos T/metabolismoRESUMO
The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and natural killer (NK) cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. Screening of our kinase-preferred collection identified aminoquinazoline 1 as a potent, nonselective inhibitor of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminoquinazolines possessing in vitro mechanism-based potency. Optimized, orally bioavailable compounds 32 and 47 exhibit anti-inflammatory activity (ED(50) of 22 and 11 mg/kg, respectively) in the anti-CD3-induced production of interleukin-2 (IL-2) in mice.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Benzamidas/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Quinazolinas/síntese química , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Benzamidas/química , Benzamidas/farmacologia , Disponibilidade Biológica , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Técnicas In Vitro , Interleucina-2/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Quinazolinas/química , Quinazolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and NK cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the synthesis, structure-activity relationships, and pharmacological characterization of 2-aminopyrimidine carbamates, a new class of compounds with potent and selective inhibition of Lck. The most promising compound of this series, 2,6-dimethylphenyl 2-((3,5-bis(methyloxy)-4-((3-(4-methyl-1-piperazinyl)propyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)carbamate (43) exhibits good activity when evaluated in in vitro assays and in an in vivo model of T cell activation.
Assuntos
Aminopiridinas/síntese química , Anti-Inflamatórios/síntese química , Carbamatos/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Pirimidinas/síntese química , Administração Oral , Aminopiridinas/química , Aminopiridinas/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Disponibilidade Biológica , Carbamatos/química , Carbamatos/farmacologia , Cristalografia por Raios X , Humanos , Técnicas In Vitro , Células Jurkat , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Estrutura Molecular , Pirimidinas/química , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
The L51S mutation in the D10.G4.1 TCR alpha-chain reduces the affinity of the TCR to its ligand by affecting the interactions among the TCR, the beta-chain of I-A(k), and the bound peptide. We show that this mutation drives the generation of a pool of memory CD44(high)CD62L(neg)CD45RB(neg) CD4 TCR transgenic T cells. Their activation threshold is low, such that they proliferate in response to lower concentrations of agonist peptides than naive L51S CD4 T cells. Unlike effector memory CD4 T cells, however, they lack immediate effector function in response to TCR stimulation. These cells express IL-2R alpha only after culture with specific peptide. Although they can be recovered from lymph nodes, the majority lack the expression of the lymph node homing receptor CCR7. When these cells receive a second TCR stimulation in vitro, they differentiate into potent Th2-like effector cells, producing high levels of IL-4 at doses of agonist peptide too low to stimulate cytokine release from similarly differentiated naive L51S CD4 T cells. Having these properties, the L51S TCR transgenic memory CD4 T cells cannot be classified as either strict central memory or effector memory, but, rather, as a pool of memory T cells containing effector memory precursors.