RESUMO
Bloom's syndrome is an autosomal recessive genome-instability disorder associated with a predisposition to cancer, premature aging and developmental abnormalities. It is caused by mutations that inactivate the DNA helicase activity of the BLM protein or nullify protein expression. The BLM helicase has been implicated in the alternative lengthening of telomeres (ALT) pathway, which is essential for the limitless replication of some cancer cells. This pathway is used by 10-15% of cancers, where inhibitors of BLM are expected to facilitate telomere shortening, leading to apoptosis or senescence. Here, the crystal structure of the human BLM helicase in complex with ADP and a 3'-overhang DNA duplex is reported. In addition to the helicase core, the BLM construct used for crystallization (residues 640-1298) includes the RecQ C-terminal (RQC) and the helicase and ribonuclease D C-terminal (HRDC) domains. Analysis of the structure provides detailed information on the interactions of the protein with DNA and helps to explain the mechanism coupling ATP hydrolysis and DNA unwinding. In addition, mapping of the missense mutations onto the structure provides insights into the molecular basis of Bloom's syndrome.
Assuntos
Difosfato de Adenosina/metabolismo , RecQ Helicases/química , RecQ Helicases/metabolismo , Síndrome de Bloom/genética , Domínio Catalítico , Cristalografia por Raios X , Humanos , Hidrólise , Modelos Moleculares , Mutação , Ácidos Nucleicos Heteroduplexes , Conformação Proteica , RecQ Helicases/genéticaRESUMO
Here we report a comprehensive biological characterization of a potent and selective small-molecule inhibitor of the DNA damage response (DDR) kinase ATR. We show a profound synthetic lethal interaction between ATR and the ATM-p53 tumor suppressor pathway in cells treated with DNA-damaging agents and establish ATR inhibition as a way to transform the outcome for patients with cancer treated with ionizing radiation or genotoxic drugs.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ligação a DNA/deficiência , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/deficiência , Pirazinas/farmacologia , Sulfonas/farmacologia , Proteína Supressora de Tumor p53/deficiência , Proteínas Supressoras de Tumor/deficiência , Animais , Antineoplásicos/química , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Estrutura Molecular , Proteínas Serina-Treonina Quinases/genética , Pirazinas/química , Sulfonas/química , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
DNA-damaging agents are among the most frequently used anticancer drugs. However, they provide only modest benefit in most cancers. This may be attributed to a genome maintenance network, the DNA damage response (DDR), that recognizes and repairs damaged DNA. ATR is a major regulator of the DDR and an attractive anticancer target. Herein, we describe the discovery of a series of aminopyrazines with potent and selective ATR inhibition. Compound 45 inhibits ATR with a K(i) of 6 nM, shows >600-fold selectivity over related kinases ATM or DNA-PK, and blocks ATR signaling in cells with an IC(50) of 0.42 µM. Using this compound, we show that ATR inhibition markedly enhances death induced by DNA-damaging agents in certain cancers but not normal cells. This differential response between cancer and normal cells highlights the great potential for ATR inhibition as a novel mechanism to dramatically increase the efficacy of many established drugs and ionizing radiation.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sequência de Aminoácidos , Antineoplásicos/síntese química , Domínio Catalítico , Modelos Moleculares , Dados de Sequência Molecular , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/química , Pirazinas/síntese química , Pirazinas/química , Pirazinas/farmacologia , Relação Estrutura-Atividade , Especificidade por Substrato , Sulfonas/químicaRESUMO
INTRODUCTION: Prevention of lymphocyte apoptosis by caspase inhibition has been proposed as a novel treatment approach in sepsis. However, it has not been clearly demonstrated that caspase inhibitors improve survival in sepsis models when dosed post-insult. Also, there are concerns that caspase inhibitors might suppress the immune response. Here we characterize VX-166, a broad caspase inhibitor, as a novel potential treatment for sepsis. METHODS: VX-166 was studied in a number of enzymatic and cellular assays. The compound was then tested in a murine model of endotoxic shock (lipopolysaccharide (LPS), 20 mg/kg IV) and a 10 d rat model of polymicrobial sepsis by caecal ligation and puncture (CLP). RESULTS: VX-166 showed potent anti-apoptotic activity in vitro and inhibited the release of interleukin (IL)-1beta and IL-18. In the LPS model, VX-166 administered 0, 4, 8 and 12 h post-LPS significantly improved survival in a dose-dependent fashion (P < 0.0028). In the CLP model, VX-166 continuously administered by mini-osmotic pump significantly improved survival when dosed 3 h after insult, (40% to 92%, P = 0.009). When dosed 8 h post-CLP, VX-166 improved survival from 40% to 66% (P = 0.19). Mode of action studies in the CLP model confirmed that VX-166 significantly inhibited thymic atrophy and lymphocyte apoptosis as determined by flow cytometry (P < 0.01). VX-166 reduced plasma endotoxin levels (P < 0.05), suggesting an improved clearance of bacteria from the bloodstream. Release of IL-1beta in vivo or T-cell activation in vitro were moderately affected. CONCLUSIONS: Our studies enhance the case for the use of caspase inhibitors in sepsis. VX-166 itself has promise as a therapy for the treatment of sepsis in man.
Assuntos
Caspases/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Choque Séptico/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Inibidores de Caspase , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/imunologia , Inibidores Enzimáticos/uso terapêutico , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/sangue , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Modelos Animais , Ratos , Ratos Sprague-Dawley , Choque Séptico/fisiopatologia , Sobrevida , Resultado do TratamentoRESUMO
The Aurora kinases are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumors, and lead to aneuploidy and tumorigenesis. Here we report the discovery of a highly potent and selective small-molecule inhibitor of Aurora kinases, VX-680, that blocks cell-cycle progression and induces apoptosis in a diverse range of human tumor types. This compound causes profound inhibition of tumor growth in a variety of in vivo xenograft models, leading to regression of leukemia, colon and pancreatic tumors at well-tolerated doses. Our data indicate that Aurora kinase inhibition provides a new approach for the treatment of multiple human malignancies.