RESUMO
The Hippo pathway and its downstream effectors, the YAP and TAZ transcriptional coactivators, are deregulated in multiple different types of human cancer and are required for cancer cell phenotypes in vitro and in vivo, while largely dispensable for tissue homeostasis in adult mice. YAP/TAZ and their main partner transcription factors, the TEAD1-4 factors, are therefore promising anticancer targets. Because of frequent YAP/TAZ hyperactivation caused by mutations in the Hippo pathway components NF2 and LATS2, mesothelioma is one of the prime cancer types predicted to be responsive to YAP/TAZ-TEAD inhibitor treatment. Mesothelioma is a devastating disease for which currently no effective treatment options exist. Here, we describe a novel covalent YAP/TAZ-TEAD inhibitor, SWTX-143, that binds to the palmitoylation pocket of all four TEAD isoforms. SWTX-143 caused irreversible and specific inhibition of the transcriptional activity of YAP/TAZ-TEAD in Hippo-mutant tumor cell lines. More importantly, YAP/TAZ-TEAD inhibitor treatment caused strong mesothelioma regression in subcutaneous xenograft models with human cells and in an orthotopic mesothelioma mouse model. Finally, SWTX-143 also selectively impaired the growth of NF2-mutant kidney cancer cell lines, suggesting that the sensitivity of mesothelioma models to these YAP/TAZ-TEAD inhibitors can be extended to other tumor types with aberrations in Hippo signaling. In brief, we describe a novel and specific YAP/TAZ-TEAD inhibitor that has potential to treat multiple Hippo-mutant solid tumor types.
Assuntos
Mesotelioma Maligno , Mesotelioma , Adulto , Humanos , Animais , Camundongos , Via de Sinalização Hippo , Proteínas de Sinalização YAP , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Mesotelioma/tratamento farmacológico , Mesotelioma/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Although anti-tumor activities of type I interferons (IFNs) have been recognized for decades, the molecular mechanisms contributing to clinical response remain poorly understood. The complex functions of these pleiotropic cytokines include stimulation of innate and adaptive immune responses against tumors as well as direct inhibition of tumor cells. In high-grade, Bacillus Calmette-Guérin (BCG)-unresponsive non-muscle-invasive bladder cancer, nadofaragene firadenovec, a non-replicating adenovirus administered locally to express the IFNα2b transgene, embodies a novel approach to deploy the therapeutic activity of type I IFNs while minimizing systemic toxicities. Deciphering which functions of type I IFN are required for clinical activity will bolster efforts to maximize the efficacy of nadofaragene firadenovec and other type I IFN-based therapies, and inform strategies to address resistance. As such, we characterized the phenotypic and molecular response of human bladder cancer cell lines to IFNα delivered in multiple contexts, including adenoviral delivery. We found that constitutive activation of the type I IFN signaling pathway is a biomarker for resistance to both transcriptional response and direct cytotoxic effects of IFNα. We present several genes that discriminate between sensitive and resistant tumor cells, suggesting they should be explored for utility as biomarkers in future clinical trials of type I IFN-based anti-tumor therapies.
RESUMO
Gonadotropin releasing hormone (GnRH) analogs have long been used in androgen deprivation therapy (ADT) in the treatment of prostate cancer. Chronic administration of either GnRH agonists or antagonists leads to suppression of testosterone production in the testes via either downregulation or direct blockade of the GnRH receptor in the pituitary, respectively. Chronic administration of kisspeptin analogs has more recently been shown to lead to testosterone suppression via desensitization of GnRH neurons in the hypothalamus and an optimized kisspeptin analog, TAK-448, was proven effective in a small phase 1 trial. The current study explored the hypothesis that co-administration of TAK-448 and the GnRH antagonist, degarelix, would have an additive effect on hormonal suppression, as a result of simultaneous intervention in separate steps in the same pathway. TAK-448 or degarelix were first administered individually to castrated rats in order to identify low doses capable of partial or no suppression of luteinizing hormone (LH). In the second step, combinations of the low doses of TAK-448 and degarelix were assessed in a 14â¯day study and compared to the drugs administered separately. The results showed that simultaneous intervention at the kisspeptin and GnRH receptors caused a more pronounced LH suppression than either drug alone, demonstrating an additive or potentiating effect. These results suggest that such a drug combination may hold promise as novel forms of androgen deprivation therapy in the treatment of prostate cancer.
Assuntos
Castração , Kisspeptinas/administração & dosagem , Kisspeptinas/farmacologia , Hormônio Luteinizante/antagonistas & inibidores , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Hormônio Luteinizante/sangue , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Antibody-drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576-87. ©2017 AACR.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Benzodiazepinas/uso terapêutico , Imunoconjugados/uso terapêutico , Pirróis/uso terapêutico , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Benzodiazepinas/efeitos adversos , Benzodiazepinas/farmacologia , Linhagem Celular Tumoral , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacologia , Masculino , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Pirróis/efeitos adversos , Pirróis/farmacologia , Ratos Sprague-Dawley , Moduladores de Tubulina/efeitos adversos , Moduladores de Tubulina/farmacologia , Moduladores de Tubulina/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Herein we report structure-cytotoxicity relationships for analogues of N14-desacetoxytubulyisn H 1. A novel synthetic approach toward 1 enabled the discovery of compounds with a range of activity. Calculated basicity of the N-terminus of tubulysins was shown to be a good predictor of cytotoxicity. The impact of structural modifications at the C-terminus of 1 upon cytotoxicity is also described. These findings will facilitate the development of new tubulysin analogues for the treatment of cancer.
Assuntos
Antineoplásicos/farmacologia , Oligopeptídeos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Relação Estrutura-AtividadeRESUMO
Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepared by chemical conjugation of small molecule cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the reduction of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophysical properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody molecule. We demonstrate that the chemically-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addition, to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clinical development.
Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Animais , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatografia Líquida de Alta Pressão , Cisteína/química , Desenho de Fármacos , Feminino , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/farmacocinética , Camundongos Nus , Terapia de Alvo Molecular , Estabilidade Proteica , Receptores Fc/químicaRESUMO
The G-protein coupled chemokine (C-X-C motif) receptor CXCR4 is linked to cancer, HIV, and WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome. While CXCR4 is reported to be overexpressed in multiple human cancer types and many hematological cancer cell lines, we have observed poor in vitro cell surface expression of CXCR4 in many solid tumor cell lines. We explore further the possible factors and pathways involved in regulating CXCR4 expression. Here, we showed that MEK-ERK signaling pathway and NFAT3 transcriptional factor plays a novel role in regulating CXCR4 expression. When cultured as 3D spheroids, HeyA8 ovarian tumor cells showed a dramatic increase in surface CXCR4 protein levels as well as mRNA transcripts. Furthermore, HeyA8 3D spheroids showed a decrease in phospho-ERK levels when compared to adherent cells. The treatment of adherent HeyA8 cells with an inhibitor of the MEK-ERK pathway, U0126, resulted in a significant increase in surface CXCR4 expression. Additional investigation using the PCR array assay comparing adherent to 3D spheroid showed a wide range of transcription factors being up-regulated, most notably a > 20 fold increase in NFAT3 transcription factor mRNA. Finally, chromatin immunoprecipitation (ChIP) analysis showed that direct binding of NFAT3 on the CXCR4 promoter corresponds to increased CXCR4 expression in HeyA8 ovarian cell line. Taken together, our results suggest that high phospho-ERK levels and NFAT3 expression plays a novel role in regulating CXCR4 expression.
Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Fatores de Transcrição NFATC/metabolismo , Receptores CXCR4/metabolismo , Western Blotting , Butadienos , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Cromonas , Ciclosporina/farmacologia , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Humanos , Ionomicina , Morfolinas , Fatores de Transcrição NFATC/antagonistas & inibidores , Nitrilas , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Esferoides Celulares , TacrolimoRESUMO
Antibody-drug conjugates (ADCs) combine the selectivity of a monoclonal antibody with the killing potency of a cytotoxic drug. For an antibody to function as a successful component of an ADC, it needs to bind to the target antigen on the surface of tumor cells and then be internalized by the cell. Following internalization, the ADC has to be transported to the lysosome where subsequent intracellular processing of the ADC will release the biologically active drug to exert its cytotoxic effects on tumor cells. This chapter describes some of the techniques that are currently used to determine internalization and proper intracellular trafficking of antibodies in order to select an optimal antibody for ADC therapeutics.
Assuntos
Citometria de Fluxo/métodos , Imunoconjugados/metabolismo , Imunoconjugados/uso terapêutico , Espaço Intracelular/metabolismo , Preparações Farmacêuticas/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Humanos , Imunoconjugados/química , Transporte ProteicoRESUMO
Inhibition of heat shock protein 90 (Hsp90) results in the degradation of oncoproteins that drive malignant progression, inducing cell death, making Hsp90 a target of substantial interest for cancer therapy. BIIB021 is a novel, fully synthetic inhibitor of Hsp90 that binds competitively with geldanamycin in the ATP-binding pocket of Hsp90. In tumor cells, BIIB021 induced the degradation of Hsp90 client proteins including HER-2, AKT, and Raf-1 and up-regulated expression of the heat shock proteins Hsp70 and Hsp27. BIIB021 treatment resulted in growth inhibition and cell death in cell lines from a variety of tumor types at nanomolar concentrations. Oral administration of BIIB021 led to the degradation of Hsp90 client proteins measured in tumor tissue and resulted in the inhibition of tumor growth in several human tumor xenograft models. Studies to investigate the antitumor effects of BIIB021 showed activity on both daily and intermittent dosing schedules, providing dose schedule flexibility for clinical studies. Assays measuring the HER-2 protein in tumor tissue and the HER-2 extracellular domain in plasma were used to show interdiction of the Hsp90 pathway and utility as potential biomarkers in clinical trials for BIIB021. Together, these data show that BIIB021 is a promising new oral inhibitor of Hsp90 with antitumor activity in preclinical models.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Piridinas/farmacologia , Adenina/administração & dosagem , Adenina/farmacocinética , Adenina/farmacologia , Administração Oral , Animais , Benzoquinonas/farmacologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Piridinas/administração & dosagem , Piridinas/farmacocinética , Receptor ErbB-2/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Misexpression and intracellular retention of peripheral myelin protein 22 (PMP22) is associated with hereditary neuropathies in humans, including Charcot-Marie-Tooth disease type 1A (CMT1A). Mice expressing extra copies of the human PMP22, termed C22, display morphologic and behavioral characteristics of CMT1A. In neuropathic Schwann cells, the turnover of the newly-synthesized PMP22 is decreased, leading to the formation of cytosolic protein aggregates. To aid the processing of PMP22 and alleviate the associated myelin defects, we pharmacologically stimulated the expression of protein chaperones by synthetic small-molecule inhibitors of heat shock protein 90 (HSP90). The exposure of Schwann cells to these compounds enhanced the levels of cytosolic chaperones in a time- and dose-dependent manner, with minimal cytotoxicity. Treatment of dorsal root ganglion (DRG) explants from neuropathic mice improved myelin formation and the processing of PMP22. These results warrant further studies with HSP90 inhibitors as potential therapeutic candidates for hereditary demyelinating neuropathies.
Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/fisiologia , Resposta ao Choque Térmico/efeitos dos fármacos , Resposta ao Choque Térmico/fisiologia , Bainha de Mielina/fisiologia , Animais , Animais Recém-Nascidos , Bovinos , Células Cultivadas , Doença de Charcot-Marie-Tooth/patologia , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/fisiopatologia , Modelos Animais de Doenças , Feminino , Proteínas de Choque Térmico HSP90/biossíntese , Resposta ao Choque Térmico/genética , Camundongos , Camundongos Transgênicos , Proteínas da Mielina/fisiologia , Bainha de Mielina/patologia , Gravidez , RatosRESUMO
Heat shock protein 90 (Hsp90) is a molecular chaperone protein implicated in stabilizing the conformation and maintaining the function of many cell-signaling proteins. Many oncogenic proteins are more dependent on Hsp90 in maintaining their conformation, stability, and maturation than their normal counterparts. Furthermore, recent data show that Hsp90 exists in an activated form in malignant cells but in a latent inactive form in normal tissues, suggesting that inhibitors selective for the activated form could provide a high therapeutic index. Hence, Hsp90 is emerging as an exciting new target for the treatment of cancer. We now report on a novel series of 2-amino-6-halopurine Hsp90 inhibitors exemplified by 2-amino-6-chloro-9-(4-iodo-3,5-dimethylpyridin-2-ylmethyl)purine (30). These highly potent inhibitors (IC50 of 30 = 0.009 microM in a HER-2 degradation assay) also display excellent antiproliferative activity against various tumor cell lines (IC50 of 30 = 0.03 microM in MCF7 cells). Moreover, this class of inhibitors shows higher affinity for the activated form of Hsp90 compared to our earlier 8-sulfanylpurine Hsp90 inhibitor series. When administered orally to mice, these compounds exhibited potent tumor growth inhibition (>80%) in an N87 xenograft model, similar to that observed with 17-allylamino-17-desmethoxygeldanamycin (17-AAG), which is a compound currently in phase I/II clinical trials.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/síntese química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Purinas/síntese química , Piridinas/síntese química , Adenina/síntese química , Adenina/química , Adenina/farmacologia , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Purinas/química , Purinas/farmacologia , Piridinas/química , Piridinas/farmacologia , Receptor ErbB-2/metabolismo , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
A primary pathologic component of Alzheimer's disease (AD) is the formation of neurofibrillary tangles composed of hyperphosphorylated tau (p-tau). Expediting the removal of these p-tau species may be a relevant therapeutic strategy. Here we report that inhibition of Hsp90 led to decreases in p-tau levels independent of heat shock factor 1 (HSF1) activation. A critical mediator of this mechanism was carboxy terminus of Hsp70-interacting protein (CHIP), a tau ubiquitin ligase. Cochaperones were also involved in Hsp90-mediated removal of p-tau, while those of the mature Hsp90 refolding complex prevented this effect. This is the first demonstration to our knowledge that blockade of the refolding pathway promotes p-tau turnover through degradation. We also show that peripheral administration of a novel Hsp90 inhibitor promoted selective decreases in p-tau species in a mouse model of tauopathy, further suggesting a central role for the Hsp90 complex in the pathogenesis of tauopathies. When taken in the context of known high-affinity Hsp90 complexes in affected regions of the AD brain, these data implicate a central role for Hsp90 in the development of AD and other tauopathies and may provide a rationale for the development of novel Hsp90-based therapeutic strategies.
Assuntos
Doença de Alzheimer/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Tauopatias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Barreira Hematoencefálica/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Fatores de Transcrição de Choque Térmico , Humanos , Masculino , Camundongos , Fosforilação , Dobramento de Proteína , RNA Interferente Pequeno/farmacologia , Serina/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The heat-shock response (HSR), a highly conserved cellular response, is characterized by rapid expression of heat-shock proteins (HSPs), and inhibition of other synthetic activities. The HSR can attenuate inflammatory responses, via suppression of transcription factor activation. A HSR can be induced pharmacologically by HSP90 inhibitors, through activation of the transcription factor Heat Shock Factor 1 (HSF1). In the present study we characterized the effects of 17-allylamino-17-demethoxygeldanamycin (17-AAG), a less toxic derivative of the naturally occurring HSP90 inhibitor geldanamycin, on glial inflammatory responses and the development of experimental autoimmune encephalomyelitis. In primary enriched glial cultures, 17-AAG dose dependently reduced lipopolysaccharide-dependent expression and activity of inducible nitric oxide synthase, attenuated interleukin (IL)-1beta expression and release, increased inhibitor of kappaB protein levels, and induced HSP70 expression. 17-AAG administration to mice immunized with myelin oligodendrocyte glycoprotein peptide prevented disease onset when given at an early time, and reduced clinical symptoms when given during ongoing disease. T cells from treated mice showed a reduced response to immunogen re-stimulation, and 17-AAG reduced CD3- and CD28-dependent IL-2 production. Together, these data suggest that HSP90 inhibitors could represent a new approach for therapeutic intervention in autoimmune diseases such as multiple sclerosis.
Assuntos
Benzoquinonas/farmacologia , Encefalite/tratamento farmacológico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Gliose/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Animais , Animais Recém-Nascidos , Anti-Inflamatórios/farmacologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/fisiopatologia , Modelos Animais de Doenças , Encefalite/imunologia , Encefalite/fisiopatologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Inibidores Enzimáticos/farmacologia , Feminino , Gliose/imunologia , Gliose/fisiopatologia , Proteínas de Choque Térmico HSP72/efeitos dos fármacos , Proteínas de Choque Térmico HSP72/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas I-kappa B/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Imunossupressores/farmacologia , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/metabolismo , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Sprague-Dawley , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Resultado do TratamentoRESUMO
We report on the discovery of benzo- and pyridino- thiazolothiopurines as potent heat shock protein 90 inhibitors. The benzothiazole moiety is exceptionally sensitive to substitutions on the aromatic ring with a 7'-substituent essential for activity. Some of these compounds exhibit low nanomolar inhibition activity in a Her-2 degradation assay (28-150 nM), good aqueous solubility, and oral bioavailability profiles in mice. In vivo efficacy experiments demonstrate that compounds of this class inhibit tumor growth in an N87 human colon cancer xenograft model via oral administration as shown with compound 37 (8-(7-chlorobenzothiazol-2-ylsulfanyl)-9-(2-cyclopropylamino-ethyl)-9H- purin-6-ylamine).
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Purinas/farmacologia , Compostos de Sulfidrila/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Nus , Estrutura Molecular , Purinas/administração & dosagem , Purinas/química , Estereoisomerismo , Relação Estrutura-Atividade , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Advanced-stage neuroblastomas are often resistant to chemotherapy. Heat shock protein (Hsp) 90 is a molecular chaperone that maintains the stability of important signal transduction proteins. We have previously reported that geldanamycin (GA), an Hsp90 inhibitor, decreases Raf-1 and Akt protein expressions and induces apoptosis in neuroblastoma cells. We sought to determine the in vivo effects of Hsp90 inhibitor compounds on human neuroblastomas. MATERIALS AND METHODS: Human neuroblastoma (LAN-1 and SK-N-SH) xenografts (4-mm3 tumor implants) were placed in the flanks of athymic nude mice. The mice received either Hsp90 inhibitors (17-AAG or EC5) or vehicle (control). The tumor dimensions were measured twice weekly. Proteins were extracted for Western immunoblotting. RESULTS: Hsp90 inhibitor compounds significantly blocked both LAN-1 and SK-N-SH neuroblastoma growth in vivo. Drug-treated tumors showed decreases in Raf-1 and cleaved PARP expressions. CONCLUSION: Hsp90 inhibitors may prove to be important novel therapeutic agents for patients with advanced-stage neuroblastoma who fail to respond to current treatment regimens.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Rifabutina/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Benzoquinonas , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Lactamas Macrocíclicas , Masculino , Camundongos , Camundongos Nus , Neuroblastoma/patologia , Rifabutina/farmacologia , Transplante Heterólogo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neurofibrillary tangles (NFTs) are a characteristic neuropathological feature of Alzheimer's disease (AD), and molecular chaperones appear to be involved in the removal of disease-associated hyperphosphorylated tau, a primary component of NFTs. Here, novel HSP90 inhibitors were used to examine the impact of chaperone elevation on clearance of different tau species in transfected cells using a unique quantitative assay. The HSP90 inhibitors reduced levels of tau phosphorylated at proline-directed Ser/Thr sites (pS202/T205, pS396/S404) and conformationally altered (MC-1) tau species, an epitope that is immeasurable by standard Western blot techniques. The selective clearance of these phospho-tau species and MC-1 tau was mediated via the proteasome, while lysosomal-mediated tau degradation seems to lack specificity for certain tau species, suggesting a more general role in total tau removal. Interestingly, tau phosphorylated at S262/S356 within the tau microtubule binding domain was minimally affected by chaperone induction. Overall, our data show that chaperone induction results in the selective clearance of specific phospho-tau and conformationally altered tau species mediated by the proteasome; however, the apparent stability of pS262/S356 tau may also explain why MARK is able to regulate normal tau function yet still be linked to the initiation of pathogenic tau hyperphosphorylation in AD.
Assuntos
Proteínas de Choque Térmico/metabolismo , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Prolina/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Cricetinae , Proteínas de Choque Térmico/antagonistas & inibidores , Humanos , Camundongos , Neurônios/metabolismo , FosforilaçãoRESUMO
Orally active Hsp90 inhibitors are of interest as potential chemotherapeutic agents. Recently, fully synthetic 8-benzyladenines and 8-sulfanyladenines such as 4 were disclosed as Hsp90 inhibitors, but these compounds are not water soluble and consequently have unacceptably low oral bioavailabilities. We now report that water-solubility can be achieved by inserting an amino functionality in the N(9) side chain. This results in compounds that are potent, soluble in aqueous media, and orally bioavailable. In an HER-2 degradation assay, the highest potency was achieved with the neopentylamine 42 (HER-2 IC(50) = 90 nM). In a murine tumor xenograft model (using the gastric cancer cell line N87), the H(3)PO(4) salts of the amines 38, 39, and 42 induced tumor growth inhibition when administered orally at 200 mg/kg/day. The amines 38, 39, and 42 are the first Hsp90 inhibitors shown to inhibit tumor growth upon oral dosage.
Assuntos
Adenina/análogos & derivados , Antineoplásicos/síntese química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Purinas/síntese química , Adenina/síntese química , Adenina/química , Adenina/farmacologia , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Feminino , Camundongos , Camundongos Nus , Purinas/química , Purinas/farmacologia , Solubilidade , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Therapeutic development for Alzheimer's disease has largely focused on the removal of beta amyloid because of its suggested role in the primary agent in initiating the disease process. However, with the recent discovery of mutations that result as pathologic buildup of tau in the absence of amyloid pathology, tau is beginning to be recognized as a potential target for drug discovery. We have developed a high-throughput drug screening method that allows for direct intracellular quantitation of tau protein species, enabling the fast, reliable detection of these changes. We have identified a family of small, blood brain barrier penetrant heat shock protein 90 inhibitors that significantly reduce tau protein levels in vitro. Western blot analysis demonstrated a clear inverse correlation between the tau levels and the increase in HSP27, HSP40 and HSP90. Modifications to this assay will further allow the specific analysis of pathologically relevant species. Using this assay, we have demonstrated that a class of HSP90 inhibitors is able to significantly lower intracellular tau levels most likely through induction of a heat shock response.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas tau/análise , Proteínas tau/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/instrumentação , Proteínas de Choque Térmico HSP90/metabolismo , HumanosRESUMO
The zeta-associated protein of 70 kDa (ZAP-70) is expressed in patients with aggressive chronic lymphocytic leukemia (CLL). We found that ZAP-70+ CLL cells expressed activated heat-shock protein 90 (Hsp90) with high binding affinity for Hsp90 inhibitors, such as 17-allyl-amino-demethoxy-geldanamycin (17-AAG), whereas normal lymphocytes or ZAP-70- CLL cells expressed nonactivated Hsp90. Activated Hsp90 bound and stabilized ZAP-70, which behaved like an Hsp90 client protein only in CLL cells. Treatment with Hsp90 inhibitors such as 17-AAG and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) induced ZAP-70 degradation and apoptosis in CLL cells but not in T cells, and also impaired B-cell receptor signaling in leukemia cells. Transduction of ZAP-70- CLL cells with an adenovirus encoding ZAP-70 activated Hsp90 and specifically rendered the leukemia cells sensitive to 17-AAG. These data indicate that Hsp90 is necessary for ZAP-70 expression and activity; that ZAP-70 is unique among Hsp90 clients, in that its chaperone-dependency is conditional on the cell type in which it is expressed; and also that ZAP-70 is required for cell survival and signaling in CLL. Additionally, ZAP-70 expression in CLL cells confers markedly heightened sensitivity to 17-AAG or 17-DMAG, suggesting that these or other Hsp90 inhibitors could be valuable therapeutically in patients with aggressive CLL.
Assuntos
Apoptose , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/terapia , Adenoviridae/genética , Adenoviridae/metabolismo , Adulto , Idoso , Antibióticos Antineoplásicos/farmacologia , Benzoquinonas , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Clonagem Molecular , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Óperon Lac , Lactamas Macrocíclicas , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucócitos Mononucleares/citologia , Linfócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Prognóstico , Ligação Proteica , Quinonas/farmacologia , Receptores de Antígenos de Linfócitos B/metabolismo , Rifabutina/análogos & derivados , Rifabutina/farmacologia , Transdução de Sinais , Linfócitos T/metabolismo , Fatores de TempoRESUMO
The widely-expressed molecular chaperone heat shock protein 90 (Hsp90) regulates several important cellular processes via its' repertoire of 'client' proteins. Signal transduction pathways controlled by Hsp90 contribute to all major components of the malignant phenotype, so Hsp90 inhibitors are under investigation as anticancer agents. Since Hsp90 is also expressed at high levels in many normal tissues, it was unclear why Hsp90 inhibitors such as 17-allylamino-geldanamycin (17-AAG) have selective antitumor activity in animals and are well tolerated clinically. Recent findings indicate that Hsp90 is largely latent in unstressed normal cells, but tumor Hsp90 becomes completely utilized during malignant progression, resulting in an activation-dependent conformational shift that radically increases 17-AAG binding affinity in cancer cells. In this article, the implications of this discovery are discussed, with particular reference to cell cycle regulation in normal and malignant cells, and the consequences of inducing cell cycle arrest with Hsp90 inhibitors.