RESUMO
An effort aimed at exploring structural diversity in the N-pyrazole-N'-naphthylurea class of p38 kinase inhibitors led to the synthesis and characterization of N-phenyl-N'-naphthylureas. Examples of these compounds displayed excellent inhibition of TNF-alpha production in vitro, as well as efficacy in a mouse model of lipopolysaccharide induced endotoxemia. In addition, perspective is provided on the role of a sulfonamide functionality in defining inhibitor potency.
Assuntos
2-Naftilamina/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Ureia/análogos & derivados , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , 2-Naftilamina/química , Animais , Química Orgânica/métodos , Química Farmacêutica/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Concentração Inibidora 50 , Lipopolissacarídeos/metabolismo , Camundongos , Modelos Químicos , Estrutura Molecular , Fator de Necrose Tumoral alfa/metabolismo , Ureia/químicaRESUMO
Integration of computational methods, X-ray crystallography, and structure-activity relationships will be disclosed, which lead to a new class of p38 inhibitors that bind to p38 MAP kinase in a Phe out conformation.
Assuntos
Modelos Moleculares , Inibidores de Proteínas Quinases/síntese química , Sulfonamidas/síntese química , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/química , Animais , Sítios de Ligação , Disponibilidade Biológica , Simulação por Computador , Cristalografia por Raios X , Desenho de Fármacos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Niacinamida/análogos & derivados , Niacinamida/síntese química , Niacinamida/química , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Tiofenos/síntese química , Tiofenos/química , Tiofenos/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossínteseRESUMO
We have previously shown that heat shock protein (Hsp) 27 or its upstream activator p38 mitogen-activated protein kinase (MAPK) confers resistance to bortezomib and dexamethasone (Dex) in multiple myeloma (MM) cells. This study examined anti-MM activity of a novel p38 MAPK inhibitor, BIRB 796, alone and in combination with conventional and novel therapeutic agents. BIRB 796 blocked baseline and bortezomib-triggered upregulation of p38 MAPK and Hsp27 phosphorylation, thereby enhancing cytotoxicity and caspase activation. The Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin (17-AAG) upregulated protein expression and phosphorylation of Hsp27; conversely, BIRB 796 inhibited this phosphorylation and enhanced 17-AAG-induced cytotoxicity. Importantly, BIRB 796 inhibited Hsp27 phosphorylation induced by 17-AAG plus bortezomib, thereby enhancing cytotoxicity. In bone marrow stromal cells (BMSC), BIRB 796 inhibited phosphorylation of p38 MAPK and secretion of interleukin-6 (IL-6) and vascular endothelial growth factor triggered by either tumour necrosis factor-alpha or tumour growth factor-beta1. BIRB 796 also inhibited IL-6 secretion induced in BMSCs by adherence to MM cells, thereby inhibiting tumour cell proliferation. These studies therefore suggest that BIRB 796 overcomes drug-resistance in the BM microenvironment, providing the framework for clinical trials of a p38 MAPK inhibitor, alone and in combination with bortezomib, Hsp90 inhibitor, or Dex, to improve patient outcome in MM.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Naftalenos/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos Hormonais/uso terapêutico , Apoptose/efeitos dos fármacos , Benzoquinonas/uso terapêutico , Ácidos Borônicos/uso terapêutico , Bortezomib , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Testes Imunológicos de Citotoxicidade , Dexametasona/uso terapêutico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Immunoblotting/métodos , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lactamas Macrocíclicas/uso terapêutico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Naftalenos/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Pirazinas/uso terapêutico , Pirazóis/farmacologia , Células-Tronco , Fator de Crescimento Transformador beta/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We report on the structure-activity relationships (SAR) of 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl]urea (BIRB 796), an inhibitor of p38alpha MAP kinase which has advanced into human clinical trials for the treatment of autoimmune diseases. Thermal denaturation was used to establish molecular binding affinities for this class of p38alpha inhibitors. The tert-butyl group remains a critical binding element by occupying a lipophilic domain in the kinase which is exposed upon rearrangement of the activation loop. An aromatic ring attached to N-2 of the pyrazole nucleus provides important pi-CH(2) interactions with the kinase. The role of groups attached through an ethoxy group to the 4-position of the naphthalene and directed into the ATP-binding domain is elucidated. Pharmacophores with good hydrogen bonding potential, such as morpholine, pyridine, and imidazole, shift the melting temperature of p38alpha by 16-17 degrees C translating into K(d) values of 50-100 pM. Finally, we describe several compounds that potently inhibit TNF-alpha production when dosed orally in mice.
Assuntos
Inibidores Enzimáticos/síntese química , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Naftalenos/síntese química , Pirazóis/síntese química , Ureia/análogos & derivados , Ureia/síntese química , Animais , Linhagem Celular , Cristalografia por Raios X , Inibidores Enzimáticos/química , Ensaio de Imunoadsorção Enzimática , Calefação , Humanos , Técnicas In Vitro , Ligantes , Lipopolissacarídeos/farmacologia , Camundongos , Proteína Quinase 14 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/química , Naftalenos/química , Ligação Proteica , Desnaturação Proteica , Pirazóis/química , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Ureia/químicaRESUMO
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.
Assuntos
Inibidores Enzimáticos/síntese química , Imidazóis/síntese química , Isoquinolinas/síntese química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/antagonistas & inibidores , Animais , Anticorpos/farmacologia , Sítios de Ligação , Complexo CD3/imunologia , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Imidazóis/química , Imidazóis/farmacologia , Interleucina-2/biossíntese , Isoquinolinas/química , Isoquinolinas/farmacologia , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Molecular , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
We report on a series of N-pyrazole, N'-aryl ureas and their mode of binding to p38 mitogen activated protein kinase. Importantly, a key binding domain that is distinct from the adenosine 5'-triphoshate (ATP) binding site is exposed when the conserved activation loop, consisting in part of Asp168-Phe169-Gly170, adopts a conformation permitting lipophilic and hydrogen bonding interactions between this class of inhibitors and the protein. We describe the correlation of the structure-activity relationships and crystallographic structures of these inhibitors with p38. In addition, we incorporated another binding pharmacophore that forms a hydrogen bond at the ATP binding site. This modification affords significant improvements in binding, cellular, and in vivo potencies resulting in the selection of 45 (BIRB 796) as a clinical candidate for the treatment of inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Inibidores Enzimáticos/síntese química , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Naftalenos/síntese química , Pirazóis/síntese química , Ureia/análogos & derivados , Ureia/síntese química , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Ligação Competitiva , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Fluorescência , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Naftalenos/química , Naftalenos/farmacologia , Ligação Proteica , Pirazóis/química , Pirazóis/farmacologia , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/biossíntese , Ureia/química , Ureia/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.