RESUMEN
A novel series of pyrrolidine heterocycles was prepared and found to show potent inhibitory activity of CCR1 binding and CCL3 mediated chemotaxis of a CCR1-expressing cell line. A potent, optimized triazole lead from this series was found to have acceptable pharmacokinetics and microsomal stability in rat and is suitable for further optimization and development.
Asunto(s)
Quimiocina CCL3/inmunología , Quimiotaxis/efectos de los fármacos , Pirrolidinas/química , Pirrolidinas/farmacología , Receptores CCR1/antagonistas & inhibidores , Animales , Línea Celular , Microsomas Hepáticos/metabolismo , Pirrolidinas/metabolismo , Pirrolidinas/farmacocinética , Ratas , Receptores CCR1/inmunología , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacocinética , Triazoles/farmacologíaRESUMEN
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.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Janus Quinasa 3/antagonistas & inhibidores , Análisis de Varianza , Animales , Artritis Experimental/metabolismo , Western Blotting , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Janus Quinasa 3/metabolismo , Ratones , Ratones Endogámicos BALB C , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
A novel class of Janus tyrosine kinase 3 (JAK3) inhibitors based on a 2-benzimidazoylpurinone core structure is described. Through substitution of the benzimidazoyl moiety and optimization of the N-9 substituent of the purinone, compound 24 was identified incorporating a chroman-based functional group. Compound 24 shows excellent kinase activity, good oral bioavailability and demonstrates efficacy in an acute mechanistic mouse model through inhibition of interleukin-2 (IL-2) induced interferon-gamma (INF-gamma) production.
Asunto(s)
Bencimidazoles/farmacología , Inhibidores Enzimáticos/farmacología , Janus Quinasa 3/antagonistas & inhibidores , Purinas/farmacología , Animales , Bencimidazoles/síntesis química , Bencimidazoles/química , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Interferón gamma/biosíntesis , Interleucina-2/antagonistas & inhibidores , Ratones , Modelos Animales , Modelos Moleculares , Estructura Molecular , Purinas/síntesis química , Purinas/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
T cell proliferation following activation is an essential aspect of the adaptive immune response. Multiple factors, such as TCR signaling, costimulation, and signals from cytokines, each contribute to determine the magnitude of T cell expansion. In this report, we examine in detail the role of Jak3/common gamma-chain-dependent cytokines in promoting cell cycle progression and proliferation of naive T cells. Using naive CD4+ T cells from Jak3-deficient mice and wild-type CD4+ T cells treated with a small molecule inhibitor of Jak3, we find that these cytokine signals are not required for proliferation; instead, they are important for the survival of activated T cells. In addition, we show that the percentage of cells entering the cell cycle and the percentage of cells in each round of cell division are comparable between Jak3-deficent and wild-type T cells. Furthermore, cell cycle progression and the regulated expression of key cell cycle proteins are independent of Jak3/common gamma-chain cytokine signals. These findings hold true over a wide range of TCR signal strengths. However, when CD28 costimulatory signals, but not TCR signals, are limiting, Jak3-dependent cytokine signals become necessary for the proliferation of naive T cells. Because CD28 signaling has been found to be dispensable for autoreactive T cell responses, these data suggest the potential for interfering with autoimmune T cell responses by inhibition of Jak3 signaling.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Ciclo Celular/inmunología , Subunidad gamma Común de Receptores de Interleucina/fisiología , Janus Quinasa 3/fisiología , Transducción de Señal/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Janus Quinasa 3/antagonistas & inhibidores , Janus Quinasa 3/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Inhibidores de Proteínas Quinasas/farmacología , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Fase de Descanso del Ciclo Celular/genética , Fase de Descanso del Ciclo Celular/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Monocyte infiltration is implicated in a variety of diseases including multiple myeloma, rheumatoid arthritis, and multiple sclerosis. C-C chemokine receptor 1 (CCR1) is a chemokine receptor that upon stimulation, particularly by macrophage inflammatory protein 1alpha (MIP-1alpha) and regulated on normal T-cell expressed and secreted (RANTES), mediates monocyte trafficking to sites of inflammation. High throughput screening of our combinatorial collection identified a novel, moderately potent CCR1 antagonist 3. The library hit 3 was optimized to the advanced lead compound 4. Compound 4 inhibited CCR1 mediated chemotaxis of monocytes with an IC(50) of 20 nM. In addition, the compound was highly selective over other chemokine receptors. It had good microsomal stability when incubated with rat and human liver microsomes and showed no significant cytochrome P450 (CYP) inhibition. Pharmacokinetic evaluation of the compound in the rat showed good oral bioavailability.
Asunto(s)
Pirrolidinas/síntesis química , Receptores CCR1/antagonistas & inhibidores , Urea/análogos & derivados , Urea/síntesis química , Administración Oral , Animales , Disponibilidad Biológica , Células CACO-2 , Permeabilidad de la Membrana Celular , Quimiotaxis de Leucocito , Inhibidores Enzimáticos del Citocromo P-450 , Humanos , Técnicas In Vitro , Isoenzimas/antagonistas & inhibidores , Microsomas Hepáticos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/fisiología , Pirrolidinas/farmacología , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Urea/farmacologíaRESUMEN
The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the alpha(v)-integrins alpha(v)beta(3) and alpha(v)beta(5) is described. High-throughput screening of an extensive series of ECLiPStrade mark compound libraries led to the identification of compound 1 as a dual inhibitor of the alpha(v)-integrins alpha(v)beta(3) and alpha(v)beta(5). Optimization of compound 1 involving, in part, introduction of two novel constraints led to the discovery of compounds 15a and 15b with reduced PSA and much improved potency for both the alpha(v)beta(3) and alpha(v)beta(5) integrins. Compounds 15a and 15b were shown to have promising activity in functional cellular assays and compound 15a also exhibited a promising Caco-2 permeability profile.
Asunto(s)
Integrina alfaVbeta3/antagonistas & inhibidores , Receptores de Vitronectina/antagonistas & inhibidores , Disponibilidad Biológica , Línea Celular , Humanos , Compuestos Macrocíclicos/farmacocinética , Compuestos Macrocíclicos/farmacología , Relación Estructura-ActividadRESUMEN
The synthesis and identification of sulfonamido-aryl ethers as potent bradykinin B1 receptor antagonists from a approximately 60,000 member encoded combinatorial library are reported. Two distinct series of compounds exhibiting different structure-activity relationships were identified in a bradykinin B1 whole-cell receptor-binding assay. Specific examples exhibit K(i) values of approximately 10nM.
Asunto(s)
Antagonistas del Receptor de Bradiquinina B1 , Éteres/síntesis química , Sulfonamidas/síntesis química , Animales , Línea Celular , Técnicas Químicas Combinatorias , Humanos , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Sulfonamidas/farmacologíaRESUMEN
The discovery and synthesis of a series of (dimethoxyphenoxy)alkylamino acetamides as orexin-2 receptor antagonists from a small-molecule combinatorial library using a high-throughput calcium mobilization functional assay (HEK293-human OX2-R cell line) is described. Active compounds show a good correlation between high-throughput single concentration screening data and measured IC(50)s. Specific examples exhibit IC(50) values of approximately 20 nM using human orexin A as the peptide agonist for the orexin-2 receptor.
Asunto(s)
Acetamidas/síntesis química , Química Farmacéutica/métodos , Péptidos y Proteínas de Señalización Intracelular/química , Neuropéptidos/química , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Neuropéptido/antagonistas & inhibidores , Acetamidas/química , Calcio/química , Línea Celular , Técnicas Químicas Combinatorias , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Mutación , Receptores de Orexina , Orexinas , TemperaturaRESUMEN
At the time of writing, there are seven marketed kinase inhibitor drugs. The first kinase inhibitor, imatinib mesilate (Gleevec, Novartis), came to market in 2001, an inhibitor of the breakpoint cluster region (BCR)/Abelson murine leukemia oncogene homolog (ABL) fusion, platelet-derived growth factor (PDGF) receptor, and c-kit kinases. The most recent kinase inhibitor to come to market, disatinib (Sprycel, Bristol-Myers Squibb), acts on c-SRC, ABL and Bruton's tyrosine kinase. To date, kinase inhibitor drugs are approved for oncology and demonstrate that it is possible to develop compounds with relative selectivity for the target kinase against the broader kinome. However, the use of kinase inhibitors in chronic inflammatory and immunologic diseases may require greater selectivity for the target kinase. This review addresses the opportunities and challenges of kinase inhibition as a therapeutic approach in chronic immune and inflammatory disease.
Asunto(s)
Enfermedades del Sistema Inmune/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Benzamidas , Enfermedad Crónica , Ensayos Clínicos como Asunto , Dasatinib , Humanos , Mesilato de Imatinib , Enfermedades del Sistema Inmune/fisiopatología , Inflamación/fisiopatología , Piperazinas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Tiazoles/farmacologíaRESUMEN
Differentiation of naive CD4+ T cells into T helper type 1 (Th1) effector cells requires both T cell receptor (TCR) signaling and cytokines such as interleukin-12 and interferon gamma (IFN-gamma). Here, we report that a third cytokine signal, mediated by the Janus family tyrosine kinase 3 (Jak3) and signal transducer and activator of transcription 5 (STAT5) pathway, is also required for Th1 cell differentiation. In the absence of Jak3-dependent signals, naive CD4+ T cells proliferate robustly but produce little IFN-gamma after Th1 cell polarization in vitro. This defect is not due to reduced activation of STAT1 or STAT4 or to impaired upregulation of the transcription factor T-bet. Instead, we find that T-bet binding to the Ifng promoter is greatly diminished in the absence of Jak3-dependent signals, correlating with a decrease in Ifng promoter accessibility and histone acetylation. These data indicate that Jak3 regulates epigenetic modification and chromatin remodeling of the Ifng locus during Th1 cell differentiation.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Ensamble y Desensamble de Cromatina , Citocinas/metabolismo , Interferón gamma/genética , Janus Quinasa 3/metabolismo , Células TH1/citología , Células TH1/metabolismo , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Citocinas/inmunología , Epigénesis Genética , Interferón gamma/inmunología , Interferón gamma/metabolismo , Ratones , Ratones Mutantes , Regiones Promotoras Genéticas , Factores de Transcripción STAT/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Células TH1/inmunologíaRESUMEN
Angiopoietins and Tie2 receptor were recently identified as an endothelial cell-specific ligand-receptor system that is critical for vascular development and postnatal pathologic angiogenesis by mediating vascular integrity. In this study, we identified a series of small-molecule Tie2 inhibitors, which blocked Ang1-induced Tie2 autophosphorylation and downstream signaling with an IC(50) value at 0.3 microM. Further optimization yields improved selectivity, aqueous solubility, microsomal stability and cytochrome P450 profile for one of the compounds (compound 7). Both compound 1 and compound 7 inhibit endothelial cell tube formation. Furthermore, in a rat model of Matrigel-induced choroidal neovascularization, compound 7 significantly diminished aberrant vessel growth. Our findings demonstrate a potential clinical benefit by specifically targeting Tie2-mediated angiogenic disorders.
Asunto(s)
Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/farmacología , Receptor TIE-2/antagonistas & inhibidores , Angiopoyetina 1/farmacología , Animales , Células Cultivadas , Coroides/irrigación sanguínea , Coroides/patología , Neovascularización Coroidal/patología , Colágeno/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Combinación de Medicamentos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Inhibidores Enzimáticos/química , Humanos , Laminina/metabolismo , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Fosforilación/efectos de los fármacos , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Solubilidad/efectos de los fármacosRESUMEN
In a high-throughput screen of four million compounds from combinatorial libraries for small-molecule modulators of the chemokine receptor CXCR3, two classes of receptor agonists, based on tetrahydroisoquinoline and piperidinyl diazepanone templates, were identified. Several of these compounds stimulated calcium flux in HEK293 cells expressing the recombinant human CXCR3 receptor with efficacies and kinetics similar to those of native ligand CXCL11/I-TAC and stimulated chemotaxis of activated human T-cells. The agonist small molecules also inhibited binding of another CXCR3 ligand, CXCL10/IP-10, to the receptor. The response to small-molecule agonists was inhibited by a CXCR3-specific small-molecule antagonist previously identified within the same combinatorial compound collection but structurally unrelated to the agonists. Remarkably, while other, non-amino acid substituents were present in the majority of the library compounds screened, the agonists from both classes contained a positively charged amino acid component, with preference for Arg>Lys, as well as a hydrophobic component.
Asunto(s)
Receptores de Quimiocina/agonistas , Bioquímica/métodos , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocinas CXC/química , Quimiocinas CXC/metabolismo , Quimiotaxis , Técnicas Químicas Combinatorias , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Ligandos , Modelos Químicos , Unión Proteica , Receptores CXCR3 , Linfocitos T/metabolismo , Tetrahidroisoquinolinas/farmacologíaRESUMEN
The identification and evaluation of aryl-[1,4]diazepane ureas as functional antagonists of the chemokine receptor CXCR3 are described. Specific examples exhibit IC(50) values of approximately 60 nM in a calcium mobilization functional assay, and dose-dependently inhibit CXCR3 functional response to CXCL11 (interferon-inducible T-cell alpha chemoattractant/I-TAC) as measured by T-cell chemotaxis, with a potency of approximately 100 nM.
Asunto(s)
Receptores de Quimiocina/antagonistas & inhibidores , Urea/química , Calcio/metabolismo , Línea Celular , Química Farmacéutica/métodos , Quimiocina CXCL11 , Quimiocinas CXC/química , Quimiotaxis , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Electrones , Humanos , Inflamación , Concentración 50 Inhibidora , Modelos Químicos , Receptores CXCR3 , Receptores de Quimiocina/química , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/química , Estereoisomerismo , Linfocitos T/citologíaRESUMEN
Secreted extracellular acid sphingomyelinase (sASM) activity has been suggested to promote atherosclerosis by enhancing subendothelial aggregation and retention of low-density lipoprotein (LDL) with resultant foam cell formation. Compounds that inhibit sASM activity, at neutral pH, may prevent lipid retention and thus would be expected to be anti-atherosclerotic. With the goal of identifying novel compounds that inhibit sASM at pH 7.4, a high-throughput screen was performed. Initial screening was run using a modification of a proven system that measures the hydrolysis of radiolabeled sphingomyelin presented in detergent micelles in a 96-well format. Separation of the radiolabeled aqueous phosphorylcholine reaction product from uncleaved sphingomyelin lipid substrate was achieved by chloroform/methanol extraction. During the screening campaign, a novel extraction procedure was developed to eliminate the use of the hazardous organic reagents. This new procedure exploited the ability of uncleaved, radiolabeled lipid substrate to interact with hydrophobic phenyl-sepharose beads. A comparison of the organic-based and the bead-based extraction sASM screening assays revealed Z' factor values ranging from 0.7 to 0.95 for both formats. In addition, both assay formats led to the identification of sub- to low micromolar inhibitors of sASM at pH 7.4 with similar IC(50) values. Subsequent studies demonstrated that both methods were also adaptable to run in a 384-well format. In contrast to the results observed at neutral pH, however, only the organic extraction assay was capable of accurately measuring sASM activity at its pH optimum of 5.0. The advantages and disadvantages of both sASM assay formats are discussed.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Esfingomielina Fosfodiesterasa/antagonistas & inhibidores , Humanos , Concentración de Iones de Hidrógeno , Micelas , Microquímica/métodosRESUMEN
Structure-activity studies on benzamide 1 obtained from library screening led to the discovery of a novel series of potent and selective glycine transporter type-2 inhibitors.