RESUMO
JNK is a stress-activated protein kinase that modulates pathways implicated in a variety of disease states. JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases. A minimal peptide region derived from JIP1 is able to inhibit JNK activity both in vitro and in cell. We report here a series of small molecules JIP1 mimics that function as substrate competitive inhibitors of JNK. One such compound, BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. In animal studies, BI-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. Our findings open the way for the development of protein kinase inhibitors targeting substrate specific docking sites, rather than the highly conserved ATP binding sites. In view of its favorable inhibition profile, selectivity, and ability to function in the cellular milieu and in vivo, BI-78D3 represents not only a JNK inhibitor, but also a promising stepping stone toward the development of an innovative class of therapeutics.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dioxanos/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/farmacologia , Animais , Ligação Competitiva , Doença Hepática Induzida por Substâncias e Drogas , Diabetes Mellitus Experimental/tratamento farmacológico , Resistência à Insulina , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Hepatopatias/prevenção & controle , Camundongos , Mimetismo Molecular , Fosforilação/efeitos dos fármacos , Ligação Proteica , Transdução de SinaisRESUMO
A series of thiadiazole derivatives has been designed as potential allosteric, substrate competitive inhibitors of the protein kinase JNK. We report on the synthesis, characterization and evaluation of a series of compounds that resulted in the identification of potent and selective JNK inhibitors targeting its JIP-1 docking site.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Tiadiazóis/síntese química , Tiadiazóis/farmacologia , Desenho de Fármacos , Células HeLa , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Tiadiazóis/químicaRESUMO
Guided by a combination of nuclear magnetic resonance binding assays and computational docking studies, we synthesized a library of 5,5' substituted Apogossypol derivatives as potent Bcl-XL antagonists. Each compound was subsequently tested for its ability to inhibit Bcl-XL in an in vitro fluorescence polarization competition assay and exert single-agent proapoptotic activity in human cancer cell lines. The most potent compound BI79D10 binds to Bcl-XL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively, and potently inhibits cell growth in the H460 human lung cancer cell line with an EC50 value of 680 nmol/L, expressing high levels of Bcl-2. BI79D10 also effectively induces apoptosis of the RS11846 human lymphoma cell line in a dose-dependent manner and shows little cytotoxicity against bax-/-bak-/- mouse embryonic fibroblast cells, in which antiapoptotic Bcl-2 family proteins lack a cytoprotective phenotype, implying that BI79D10 has little off-target effects. BI79D10 displays in vivo efficacy in transgenic mice, in which Bcl-2 is overexpressed in splenic B cells. Together with its improved plasma and microsomal stability relative to Apogossypol, BI79D10 represents a lead compound for the development of novel apoptosis-based therapies for cancer.
Assuntos
Gossipol/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Linfoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Sobrevivência Celular/efeitos dos fármacos , Feminino , Polarização de Fluorescência , Gossipol/síntese química , Gossipol/química , Gossipol/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Linfoma/metabolismo , Linfoma/patologia , Espectroscopia de Ressonância Magnética , Masculino , Proteínas de Membrana/metabolismo , Membranas Artificiais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Microssomos Hepáticos , Modelos Moleculares , Proteína de Sequência 1 de Leucemia de Células Mieloides , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Células Tumorais Cultivadas , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Proteína X Associada a bcl-2/fisiologia , Proteína bcl-X/metabolismoRESUMO
We report on the identification of a novel small molecule inhibitor of anthrax lethal factor using a high-throughput screening approach. Guided by molecular docking studies, we carried out structure-activity relationship (SAR) studies and evaluated activity and selectivity of most promising compounds in in vitro enzyme inhibition assays and cellular assays. Selected compounds were further analyzed for their in vitro ADME properties, which allowed us to select two compounds for further preliminary in vivo efficacy studies. The data provided represents the basis for further pharmacology and medicinal chemistry optimizations that could result in novel anti-anthrax therapies.
Assuntos
Antígenos de Bactérias/química , Antitoxinas/química , Antitoxinas/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/química , Sulfonamidas/química , Sulfonamidas/farmacologia , Animais , Antraz/tratamento farmacológico , Bacillus anthracis/metabolismo , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonamidas/síntese químicaRESUMO
A new series of 2-thioether-benzothiazoles has been synthesized and evaluated for JNK inhibition. The SAR studies led to the discovery of potent, allosteric JNK inhibitors with selectivity against p38.
Assuntos
Benzotiazóis/química , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Alostérica , Benzotiazóis/síntese química , Benzotiazóis/farmacologia , Simulação por Computador , Descoberta de Drogas , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We report on the synthesis and evaluation of an indazole-spin-labeled compound that was designed as an effective chemical probe for second site screening against the protein kinase JNK using NMR-based techniques. We demonstrate the utility of the derived compound in detecting and characterizing binding events at the protein kinase docking site. In addition, we report on the NMR-based design and synthesis of a bidentate compound spanning both the ATP site and the docking site. We show that the resulting compound has nanomolar affinity for JNK despite the relatively weak affinities of the individual fragments that constitute it. The approach demonstrates that targeting the docking site of protein kinases represents a valuable yet unexplored avenue to obtain potent kinase inhibitors with increased selectivity.
Assuntos
Óxidos N-Cíclicos/síntese química , Indazóis/síntese química , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Modelos Moleculares , Oligopeptídeos/síntese química , Marcadores de Spin/síntese química , Sítios de Ligação , Óxidos N-Cíclicos/química , Indazóis/química , Proteínas Quinases JNK Ativadas por Mitógeno/química , Espectroscopia de Ressonância Magnética , Oligopeptídeos/química , Ligação ProteicaRESUMO
Peptidyl-prolyl cis-trans isomerases are a group of cytosolic enzymes initially characterized by their ability to catalyze the cis-trans isomerization of peptidyl-prolyl bonds. This represents a significant event for protein folding because cis-proline introduces critical bends within the protein conformation. FK506-binding proteins (FKBPs) represent one of the three families of enzymes sharing peptidyl-prolyl cis-trans isomerase activity. Inhibitors of FKBP12, in particular, have potent neurotrophic properties both in vivo and in vitro. Here, we describe a fragment-based unbiased nuclear magnetic resonance drug discovery approach for the identification of novel classes of chemical inhibitors against FKBP12. Compared to FK506, the fragment-based FKBP12 inhibitors developed herein possess significant advantages as drug candidates.
Assuntos
Morfolinas/síntese química , Proteína 1A de Ligação a Tacrolimo/antagonistas & inibidores , Animais , Linhagem Celular , Desenho de Fármacos , Humanos , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Morfolinas/química , Morfolinas/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Ratos , Ratos Long-Evans , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Tacrolimo/química , Tacrolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/químicaRESUMO
We report comprehensive structure-activity relationship studies on a novel series of c-Jun N-terminal kinase (JNK) inhibitors. The compounds are substrate competitive inhibitors that bind to the docking site of the kinase. The reported medicinal chemistry and structure-based optimizations studies resulted in the discovery of selective and potent thiadiazole JNK inhibitors that display promising in vivo activity in mouse models of insulin insensitivity.
Assuntos
Hipoglicemiantes/síntese química , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Tiadiazóis/síntese química , Tiazóis/síntese química , Triazóis/síntese química , Fator 2 Ativador da Transcrição/metabolismo , Animais , Sítios de Ligação , Diabetes Mellitus Tipo 2/tratamento farmacológico , Desenho de Fármacos , Células HeLa , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Resistência à Insulina , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Fosforilação , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Tiadiazóis/química , Tiadiazóis/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Triazóis/química , Triazóis/farmacologiaRESUMO
Guided by nuclear magnetic resonance (NMR) binding assays and computational docking studies, a series of 5,5' substituted apogossypol derivatives was synthesized that resulted in potent pan-active inhibitors of antiapoptotic Bcl-2 family proteins. Compound 8r inhibits the binding of BH3 peptides to Bcl-X(L), Bcl-2, Mcl-1, and Bfl-1 with IC(50) values of 0.76, 0.32, 0.28, and 0.73 microM, respectively. The compound also potently inhibits cell growth of human lung cancer and BP3 human B-cell lymphoma cell lines with EC(50) values of 0.33 and 0.66 microM, respectively. Compound 8r shows little cytotoxicity against bax(-/-)bak(-/-) cells, indicating that it kills cancers cells via the intended mechanism. The compound also displays in vivo efficacy in transgenic mice in which Bcl-2 is overexpressed in splenic B-cells. Together with its improved chemical, plasma, and microsomal stability relative to compound 2 (apogossypol), compound 8r represents a promising drug lead for the development of novel apoptosis-based therapies for cancer.
Assuntos
Apoptose/efeitos dos fármacos , Gossipol/análogos & derivados , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Alquilação , Amidas/química , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Gossipol/sangue , Gossipol/química , Gossipol/metabolismo , Gossipol/farmacologia , Humanos , Cetonas/química , Espectroscopia de Ressonância Magnética , Camundongos , Microssomos/metabolismo , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Especificidade por SubstratoRESUMO
We report on a general structure- and NMR-based approach to derive druglike small molecule inhibitors of protein-protein interactions in a rapid and efficient manner. We demonstrate the utility of the approach by deriving novel and effective SMAC mimetics targeting the antiapoptotic protein X-linked inhibitor of apoptosis protein (XIAP). The XIAP baculovirus IAP repeat 3 (Bir3) domain binds directly to the N-terminal of caspase-9, thus inhibiting programmed cell death. It has been shown that in the cell this interaction can be displaced by the protein second mitochondrial activator of caspases (SMAC) and that its N-terminal tetrapeptide region (NH2-AVPI, Ala-Val-Pro-Ile) is responsible for this activity. However, because of their limited cell permeability, synthetic SMAC peptides are inefficient when tested in cultured cells, limiting their use as potential chemical tools or drug candidates against cancer cells. Hence, as an application, we report on the derivation of novel, selective, druglike, cell permeable SMAC mimics with cellular activity.