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
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
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 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.