RESUMO
For the past two decades, BTK a tyrosine kinase and member of the Tec family has been a drug target of significant interest due to its potential to selectively treat various B cell-mediated diseases such as CLL, MCL, RA, and MS. Owning to the challenges encountered in identifying drug candidates exhibiting the potency block B cell activation via BTK inhibition, the pharmaceutical industry has relied on the use of covalent/irreversible inhibitors to address this unmet medical need. Herein, we describe a medicinal chemistry campaign to identify structurally diverse reversible BTK inhibitors originating from HITS identified using a fragment base screen. The leads were optimized to improve the potency and in vivo ADME properties resulting in a structurally distinct chemical series used to develop and validate a novel in vivo CD69 and CD86 PD assay in rodents.
Assuntos
Inibidores de Proteínas Quinases , Proteínas Tirosina Quinases , Camundongos , Animais , Tirosina Quinase da Agamaglobulinemia , Inibidores de Proteínas Quinases/química , Modelos Animais de Doenças , Antígeno B7-2RESUMO
OBJECTIVE: TGF-beta plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis. METHODS AND RESULTS: The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF-beta and activin-induced Smad2/3 phosphorylation and TGF-beta-induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle alpha-actin-positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells. CONCLUSIONS: These results are the first to demonstrate the efficacy of an orally active, small-molecule ALK5/ALK4 inhibitor in a vascular fibrosis model and suggest the potential therapeutic application of these inhibitors in vascular fibrosis.
Assuntos
Compostos Azabicíclicos/farmacologia , Lesões das Artérias Carótidas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Ativinas Tipo I/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Administração Oral , Animais , Compostos Azabicíclicos/administração & dosagem , Compostos Azabicíclicos/metabolismo , Sítios de Ligação , Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/fisiopatologia , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose , Humanos , Masculino , Mioblastos de Músculo Liso/efeitos dos fármacos , Mioblastos de Músculo Liso/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor do Fator de Crescimento Transformador beta Tipo I , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Protein interacting with C kinase (PICK1) is a scaffolding protein that is present in dendritic spines and interacts with a wide array of proteins through its PDZ domain. The best understood function of PICK1 is regulation of trafficking of AMPA receptors at neuronal synapses via its specific interaction with the AMPA GluA2 subunit. Disrupting the PICK1-GluA2 interaction has been shown to alter synaptic plasticity, a molecular mechanism of learning and memory. Lack of potent, selective inhibitors of the PICK1 PDZ domain has hindered efforts at exploring the PICK1-GluA2 interaction as a therapeutic target for neurological diseases. Here, we report the discovery of PICK1 small molecule inhibitors using a structure-based drug design strategy. The inhibitors stabilized surface GluA2, reduced Aß-induced rise in intracellular calcium concentrations in cultured neurons, and blocked long term depression in brain slices. These findings demonstrate that it is possible to identify potent, selective PICK1-GluA2 inhibitors which may prove useful for treatment of neurodegenerative disorders.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Espinhas Dendríticas/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Sinapses/metabolismo , Animais , Encéfalo/patologia , Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Espinhas Dendríticas/patologia , Desenho de Fármacos , Camundongos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Proteínas Nucleares/metabolismo , Domínios PDZ , Receptores de AMPA/metabolismo , Sinapses/patologiaRESUMO
Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 A resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 A resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.
Assuntos
Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Tirosina Quinase da Agamaglobulinemia , Sequência de Aminoácidos , Cristalografia por Raios X , Dasatinibe , Ativação Enzimática , Humanos , Dados de Sequência Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/química , Pirróis/química , Tiazóis/químicaRESUMO
PEGylation of IFN-alpha has been used successfully to improve the pharmacokinetic properties and efficacy of the drug. To prepare a PEGylated form of human interferon-beta-1a (IFN-beta-1a) suitable for testing in vivo, we have synthesized 20 kDa mPEG-O-2-methylpropionaldehyde and used it to modify the N-terminal alpha-amino group of the cytokine. The PEGylated protein retained approximately 50% of the activity of the unmodified protein and had significantly improved pharmacokinetic properties following intravenous administration in rats. The clearance and volume of distribution at steady state were reduced approximately 30-fold and approximately 4-fold, respectively, resulting in a significant increase in systemic exposure as determined by the area under the curve. The elimination half-life of the PEGylated protein was approximately 13-fold greater than for the unmodified protein. The unmodified and PEGylated proteins were tested for their ability to inhibit the formation of radially oriented blood vessels entering the periphery of human SK-MEL-1 melanoma tumors in athymic nude homozygous (nu/nu) mice. In a single dose comparison study, administration of 1 x 10(6) units of unmodified IFN-beta-1a resulted in a 29% reduction in vessel number, while 1 x 10(6) units of PEGylated IFN-beta-1a resulted in a 58% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle (control)-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. In a multiple versus single dose comparison study, daily administration of 1 x 10(6) units of unmodified IFN-beta-1a for 9 days resulted in a 51% reduction in vessel number, while a single dose of 1 x 10(6) units of the PEGylated protein resulted in a 66% reduction. Both treatments resulted in statistically significant reductions in mean vessel number as compared to the vehicle-treated mice, with the PEGylated IFN-beta-1a-treated mice showing a statistically significantly greater reduction in mean vessel number as compared to the unmodified IFN-beta-1a-treated mice. Therefore, the improved pharmacokinetic properties of the modified protein translated into improved efficacy. Since unmodified IFN-beta is used for the treatment of multiple sclerosis and hepatitis C virus infection, a PEGylated form of the protein such as 20 kDa mPEG-O-2-methylpropionaldehyde-modified IFN-beta-1a may serve as a useful adjunct for the treatment of these diseases. In addition, the antiangiogenic effects of PEGylated IFN-beta-1a may be harnessed for the treatment of certain cancers, either as a sole agent or in combination with other antitumor drugs.
Assuntos
Aldeídos/uso terapêutico , Antivirais/uso terapêutico , Interferon beta/uso terapêutico , Melanoma Experimental/tratamento farmacológico , Neovascularização Patológica/prevenção & controle , Polietilenoglicóis/uso terapêutico , Aldeídos/síntese química , Aldeídos/farmacocinética , Animais , Antivirais/química , Antivirais/farmacocinética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Vírus da Encefalomiocardite/efeitos dos fármacos , Feminino , Meia-Vida , Humanos , Interferon beta-1a , Interferon beta/química , Interferon beta/farmacocinética , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/patologia , Taxa de Depuração Metabólica , Camundongos , Camundongos Nus , Transplante de Neoplasias , Polietilenoglicóis/síntese química , Polietilenoglicóis/farmacocinética , Ratos , Ratos Endogâmicos LewRESUMO
Engagement of the IFN-alphabeta receptor initiates multiple signaling cascades, including activation of the STAT. In this study, we demonstrate that IFN-alphabeta, although antiproliferative in wild-type CD4(+) or CD8(+) T cells, act as strong mitogens on their STAT1(-/-) counterparts. Furthermore, IFN-alphabeta exert little effect on apoptosis in wild-type cells, but are potent survival factors in the absence of STAT1. The antiapoptotic response in the absence of STAT1 is predominantly mediated by STAT3, and to a lesser extent by STAT5A/B. In contrast, the mitogenic IFN-alphabeta response gained through the absence of STAT1 is only marginally affected when STAT5A/B expression is also abrogated, but is completely dependent on STAT3 activation. These findings provide the first evidence for a function of STAT3 and STAT5A/B in the IFN-alphabeta response, and support a model in which the IFN-alphabeta receptor initiates both pro- and antiapoptotic responses through STAT1, and STAT3 and STAT5A/B, respectively.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Interferon Tipo I/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transativadores/fisiologia , Animais , Apoptose/genética , Apoptose/imunologia , Proliferação de Células , Sobrevivência Celular/imunologia , Células Cultivadas , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Inibidores do Crescimento/farmacologia , Inibidores do Crescimento/fisiologia , Interferon Tipo I/farmacologia , Interferon-alfa/fisiologia , Interferon beta/farmacologia , Interferon beta/fisiologia , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Proteínas do Leite/genética , Mitógenos/farmacologia , Mitógenos/fisiologia , Fase de Repouso do Ciclo Celular/genética , Fase de Repouso do Ciclo Celular/imunologia , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Fator de Transcrição STAT5 , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/citologia , Transativadores/deficiência , Transativadores/genéticaRESUMO
To identify potential new clinical uses and routes of administration for human interferon-beta-1a (IFN-beta-1a), we have developed an expression and purification procedure for the preparation of highly purified rat interferon-beta (IFN-beta) suitable for testing in rat models of human disease. An expression vector containing the rat IFN-beta signal sequence and structural gene was constructed and transfected into Chinese hamster ovary (CHO) cells. The protein was purified from CHO cell conditioned medium and purified to > 99.5% purity using standard chromatographic techniques. Analytical characterization indicated that the protein was a heavily glycosylated monomeric protein, with two of the four predicted N-glycosylation sites occupied. Analysis of the attached oligosaccharides showed them to be a complex mixture of bi-antennary, tri-antennary, and tetra-antennary structures with a predominance of sialylated tri-antennary and tetra-antennary structures. Peptide mapping, N-terminal sequencing, and mass spectrometry confirmed the identity and integrity of the purified protein. The purified protein had a specific activity of 2.1x10(8)U/mg when assayed on rat RATEC cells, which is similar in magnitude to the potencies observed for murine IFN-beta and human IFN-beta-1a assayed on murine and human cells, respectively. We also prepared an N-terminally PEGylated form of rat IFN-beta in which a 20 kDa methoxy polyethylene glycol (PEG)-propionaldehyde was attached to the N-terminal alpha-amino group of Ile-1. The PEGylated protein, which retained essentially full in vitro antiviral activity, had improved pharmacokinetic parameters in rats as compared to the unmodified protein. Both the unmodified and PEGylated forms of rat IFN-beta will be useful for testing in rat models of human disease.
Assuntos
Interferon Tipo I/metabolismo , Interferon beta/metabolismo , Oligossacarídeos/metabolismo , Polietilenoglicóis/química , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Glicosilação , Humanos , Interferon Tipo I/genética , Interferon Tipo I/isolamento & purificação , Interferon beta-1a , Interferon beta/genética , Espectrometria de Massas , Ratos , Proteínas RecombinantesRESUMO
We describe the discovery, using shape-based virtual screening, of a potent, ATP site-directed inhibitor of the TbetaRI kinase, an important and novel drug target for fibrosis and cancer. The first detailed report of a TbetaRI kinase small molecule co-complex confirms the predicted binding interactions of our small molecule inhibitor, which stabilizes the inactive kinase conformation. Our results validate shape-based screening as a powerful tool to discover useful leads against a new drug target.