RESUMO
EGFR is frequently mutated and amplified in lung adenocarcinomas sensitive to EGFR inhibitors gefitinib and erlotinib. A secondary mutation, T790M, has been associated with acquired resistance but has not been shown to be sufficient to render EGFR mutant/amplified lung cancers resistant to EGFR inhibitors. We created a model for studying acquired resistance to gefitinib by prolonged exposure of a gefitinib-sensitive lung carcinoma cell line (H3255; EGFR mutated and amplified) to gefitinib in vitro. The resulting resistant cell line acquired a T790M mutation in a small fraction of the amplified alleles that was undetected by direct sequencing and identified only by a highly sensitive HPLC-based technique. In gefitinib-sensitive lung cancer cells with EGFR mutations and amplifications, exogenous introduction of EGFR T790M effectively conferred resistance to gefitinib and continued ErbB-3/PI3K/Akt signaling when in cis to an activating mutation. Moreover, continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110alpha E545K, was sufficient to abrogate gefitinib-induced apoptosis. These findings suggest that allelic dilution of biologically significant resistance mutations may go undetected by direct sequencing in cancers with amplified oncogenes and that restoration of PI3K activation via either a T790M mutation or other mechanisms can provide resistance to gefitinib.
Assuntos
Alelos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutação de Sentido Incorreto/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gefitinibe , Amplificação de Genes , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Interferência de RNA , Receptor ErbB-3/metabolismo , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent events have created an urgent need for new therapeutic strategies to treat anthrax. We have applied a mixture-based peptide library approach to rapidly determine the optimal peptide substrate for the anthrax lethal factor (LF), a metalloproteinase with an important role in the pathogenesis of the disease. Using this approach we have identified peptide analogs that inhibit the enzyme in vitro and that protect cultured macrophages from LF-mediated cytolysis. The crystal structures of LF bound to an optimized peptide substrate and to peptide-based inhibitors provide a rationale for the observed selectivity and may be exploited in the design of future generations of LF inhibitors.
Assuntos
Antígenos de Bactérias , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Antraz/tratamento farmacológico , Bacillus anthracis/enzimologia , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Humanos , Técnicas In Vitro , Cinética , Substâncias Macromoleculares , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Biblioteca de Peptídeos , Especificidade por SubstratoRESUMO
Selection of target substrates by protein kinases is strongly influenced by the amino acid sequence surrounding the phosphoacceptor site. Identification of the preferred peptide phosphorylation motif for a given kinase permits the production of efficient peptide substrates and greatly simplifies the mapping of phosphorylation sites in protein substrates. Here we describe a combinatorial peptide library method that allows rapid generation of phosphorylation motifs for serine/threonine kinases.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Biblioteca de Peptídeos , Mapeamento de Interação de Proteínas/métodos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Análise de Sequência de Proteína/métodos , Motivos de Aminoácidos , Sítios de Ligação , Ativação Enzimática , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/análise , Especificidade por SubstratoRESUMO
Inhibitor of apoptosis proteins (IAPs) prevent apoptosis through direct inhibition of caspases. The serine protease HtrA2/Omi has an amino-terminal IAP interaction motif like that found in Reaper, which displaces IAPs from caspases, leading to enhanced caspase activity. The cell death-promoting properties of HtrA2/Omi are not only exerted through its capacity to oppose IAP inhibition of caspases but also through its integral serine protease activity. We have used peptide libraries to determine the optimal substrate sequence for cleavage by HtrA2 and also the preferred binding sequence for its PDZ domain. Using these peptides, we show that the PDZ domain of HtrA2/Omi suppresses the proteolytic activity unless it is engaged by a binding partner. Subjecting HtrA2/Omi to heat shock treatment also increases its protease activity. Unexpectedly, binding of X-linked inhibitor of apoptosis protein (XIAP) to the Reaper motif of HtrA2/Omi results in a marked increase in proteolytic activity, suggesting a new role for IAPs. When HtrA2/Omi is released from mitochondria following an apoptotic stimulus, binding to IAPs may switch their function from caspase inhibition to serine protease activation. Thus although IAP overexpression can suppress caspase activation, it could have the opposite effect on HtrA2/Omi-dependent cell death. This, together with the ability of HtrA2/Omi to degrade IAPs, may limit the overall cellular protection that can be provided by these proteins.