RESUMO
Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.
Assuntos
Antineoplásicos/farmacocinética , Benzoquinonas/farmacocinética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacocinética , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Lactamas Macrocíclicas/farmacologia , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos NusRESUMO
Four enantiopure cytisine-inspired scaffolds can be accessed via a versatile pyrrolidine template derived from a stereocontrolled [3+2] azomethine ylide-alkene cycloaddition. Differential ester protection allows for the selective formation of either a bridged bicyclic or tricyclic scaffold via pyridone cyclization. Solid-phase diversification of the pyridone scaffolds yielded a diverse library of 15,000 compounds enabling the discovery of a novel class of Bcl-2 inhibitors.
Assuntos
Alcaloides/farmacologia , Química Farmacêutica/métodos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Piridonas/síntese química , Apoptose , Azocinas/farmacologia , Desenho de Fármacos , Humanos , Cinética , Modelos Químicos , Estrutura Molecular , Mapeamento de Interação de Proteínas , Piridonas/química , Pirrolidinas/química , Quinolizinas/farmacologia , Estereoisomerismo , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/químicaRESUMO
A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between beta-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX trade mark system, uses a pair of inactive beta-galactosidase (beta-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and beta-arrestin-beta-gal fusion proteins are generated. Following ligand stimulation, beta-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the beta-gal mutant fragments. GPCR activation is measured directly by quantitating restored beta-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the beta2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The beta2-adrenergic receptor cell line was screened with the LOPAC trade mark compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.
Assuntos
Bioensaio/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Mapeamento de Interação de Proteínas/métodos , Receptores Acoplados a Proteínas G/metabolismo , beta-Galactosidase/metabolismo , Arrestinas/genética , Arrestinas/metabolismo , Bioensaio/instrumentação , Células Cultivadas , Técnicas de Química Combinatória/métodos , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/instrumentação , Genes erbB-1 , Humanos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mapeamento de Interação de Proteínas/instrumentação , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores de Interleucina-8B/efeitos dos fármacos , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , beta-Galactosidase/genéticaRESUMO
HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.
Assuntos
Benzoquinonas/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Taxoides/administração & dosagem , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Benzoquinonas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Docetaxel , Regulação para Baixo , Sinergismo Farmacológico , Humanos , Lactamas Macrocíclicas/farmacologia , Neoplasias Pulmonares/metabolismo , Masculino , Taxoides/farmacologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Phosphoinositide-3 kinase (PI3K)-δ and PI3K-γ are preferentially expressed in immune cells, and inhibitors targeting these isoforms are hypothesized to have anti-inflammatory activity by affecting the adaptive and innate immune response. We report on a potent oral PI3K-δ and PI3K-γ inhibitor (IPI-145) and characterize this compound in biochemical, cellular, and in vivo assays. These studies demonstrate that IPI-145 exerts profound effects on adaptive and innate immunity by inhibiting B and T cell proliferation, blocking neutrophil migration, and inhibiting basophil activation. We explored the therapeutic value of combined PI3K-δ and PI3K-γ blockade, and IPI-145 showed potent activity in collagen-induced arthritis, ovalbumin-induced asthma, and systemic lupus erythematosus rodent models. These findings support the hypothesis that inhibition of immune function can be achieved through PI3K-δ and PI3K-γ blockade, potentially leading to significant therapeutic effects in multiple inflammatory, autoimmune, and hematologic diseases.
Assuntos
Artrite/tratamento farmacológico , Asma/tratamento farmacológico , Modelos Animais de Doenças , Isoquinolinas/farmacologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Purinas/farmacologia , Animais , Artrite/induzido quimicamente , Artrite/imunologia , Asma/induzido quimicamente , Asma/imunologia , Colágeno Tipo II , Relação Dose-Resposta a Droga , Feminino , Humanos , Isoquinolinas/química , Lúpus Eritematoso Sistêmico/imunologia , Estrutura Molecular , Ovalbumina , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Purinas/química , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.