RESUMO
Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.
Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Melanoma/imunologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Humanos , Tolerância Imunológica/efeitos dos fármacos , Masculino , Melanoma/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Mieloides/enzimologia , Metástase Neoplásica/tratamento farmacológico , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Macrophages play critical, but opposite, roles in acute and chronic inflammation and cancer. In response to pathogens or injury, inflammatory macrophages express cytokines that stimulate cytotoxic T cells, whereas macrophages in neoplastic and parasitic diseases express anti-inflammatory cytokines that induce immune suppression and may promote resistance to T cell checkpoint inhibitors. Here we show that macrophage PI 3-kinase γ controls a critical switch between immune stimulation and suppression during inflammation and cancer. PI3Kγ signalling through Akt and mTor inhibits NFκB activation while stimulating C/EBPß activation, thereby inducing a transcriptional program that promotes immune suppression during inflammation and tumour growth. By contrast, selective inactivation of macrophage PI3Kγ stimulates and prolongs NFκB activation and inhibits C/EBPß activation, thus promoting an immunostimulatory transcriptional program that restores CD8+ T cell activation and cytotoxicity. PI3Kγ synergizes with checkpoint inhibitor therapy to promote tumour regression and increased survival in mouse models of cancer. In addition, PI3Kγ-directed, anti-inflammatory gene expression can predict survival probability in cancer patients. Our work thus demonstrates that therapeutic targeting of intracellular signalling pathways that regulate the switch between macrophage polarization states can control immune suppression in cancer and other disorders.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Tolerância Imunológica/imunologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Células Cultivadas , Classe Ib de Fosfatidilinositol 3-Quinase/deficiência , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Feminino , Humanos , Inflamação/imunologia , Ativação Linfocitária , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neoplasias/imunologia , Neoplasias/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Serina-Treonina Quinases TOR/metabolismo , Evasão Tumoral/imunologiaRESUMO
A hallmark of prostate cancer progression is dysregulation of lipid metabolism via overexpression of fatty acid synthase (FASN), a key enzyme in de novo fatty acid synthesis. Metastatic castration-resistant prostate cancer (mCRPC) develops resistance to inhibitors of androgen receptor (AR) signaling through a variety of mechanisms, including the emergence of the constitutively active AR variant V7 (AR-V7). Here, we developed an FASN inhibitor (IPI-9119) and demonstrated that selective FASN inhibition antagonizes CRPC growth through metabolic reprogramming and results in reduced protein expression and transcriptional activity of both full-length AR (AR-FL) and AR-V7. Activation of the reticulum endoplasmic stress response resulting in reduced protein synthesis was involved in IPI-9119-mediated inhibition of the AR pathway. In vivo, IPI-9119 reduced growth of AR-V7-driven CRPC xenografts and human mCRPC-derived organoids and enhanced the efficacy of enzalutamide in CRPC cells. In human mCRPC, both FASN and AR-FL were detected in 87% of metastases. AR-V7 was found in 39% of bone metastases and consistently coexpressed with FASN. In patients treated with enzalutamide and/or abiraterone FASN/AR-V7 double-positive metastases were found in 77% of cases. These findings provide a compelling rationale for the use of FASN inhibitors in mCRPCs, including those overexpressing AR-V7.
Assuntos
Lipogênese , Proteínas de Neoplasias/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Humanos , Masculino , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CD39 (ENTPD1) is a key enzyme responsible for degradation of extracellular ATP and is upregulated in the tumor microenvironment (TME). Extracellular ATP accumulates in the TME from tissue damage and immunogenic cell death, potentially initiating proinflammatory responses that are reduced by the enzymatic activity of CD39. Degradation of ATP by CD39 and other ectonucleotidases (e.g., CD73) results in extracellular adenosine accumulation, constituting an important mechanism for tumor immune escape, angiogenesis induction, and metastasis. Thus, inhibiting CD39 enzymatic activity can inhibit tumor growth by converting a suppressive TME to a proinflammatory environment. SRF617 is an investigational, anti-CD39, fully human IgG4 Ab that binds to human CD39 with nanomolar affinity and potently inhibits its ATPase activity. In vitro functional assays using primary human immune cells demonstrate that inhibiting CD39 enhances T-cell proliferation, dendritic cell maturation/activation, and release of IL-1ß and IL-18 from macrophages. In vivo, SRF617 has significant single-agent antitumor activity in human cell line-derived xenograft models that express CD39. Pharmacodynamic studies demonstrate that target engagement of CD39 by SRF617 in the TME inhibits ATPase activity, inducing proinflammatory mechanistic changes in tumor-infiltrating leukocytes. Syngeneic tumor studies using human CD39 knock-in mice show that SRF617 can modulate CD39 levels on immune cells in vivo and can penetrate the TME of an orthotopic tumor, leading to increased CD8+ T-cell infiltration. Targeting CD39 is an attractive approach for treating cancer, and, as such, the properties of SRF617 make it an excellent drug development candidate.
Assuntos
Imunoglobulina G , Ativação Linfocitária , Humanos , Animais , Camundongos , Anticorpos Monoclonais , Adenosina Trifosfatases , Trifosfato de AdenosinaRESUMO
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
The neonatal Fc receptor FcRn provides IgG molecules with their characteristically long half-lives in vivo by protecting them from intracellular catabolism and then returning them to the extracellular space. Other investigators have demonstrated that mice lacking FcRn are protected from induction of various autoimmune diseases, presumably because of the accelerated catabolism of pathogenic IgGs in the animals. Therefore, targeting FcRn with a specific inhibitor may represent a unique approach for the treatment of autoimmune disease or other diseases where the reduction of pathogenic IgG will have a therapeutic benefit. Using phage display peptide libraries, we screened for ligands that bound to human FcRn (hFcRn) and discovered a consensus peptide sequence that binds to hFcRn and inhibits the binding of human IgG (hIgG) in vitro. Chemical optimization of the phage-identified sequences yielded the 26-amino acid peptide dimer SYN1436, which is capable of potent in vitro inhibition of the hIgG-hFcRn interaction. Administration of SYN1436 to mice transgenic for hFcRn induced an increase in the rate of catabolism of hIgG in a dose-dependent manner. Treatment of cynomolgus monkeys with SYN1436 led to a reduction of IgG by up to 80% without reducing serum albumin levels that also binds to FcRn. SYN1436 and related peptides thus represent a previously uncharacterized family of potential therapeutic agents for the treatment of humorally mediated autoimmune and other diseases.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Macaca fascicularis/imunologia , Peptídeos/imunologia , Receptores Fc/antagonistas & inibidores , Receptores Fc/imunologia , Albuminas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Imunoglobulina A/imunologia , Imunoglobulina M/imunologia , Macaca fascicularis/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Solubilidade , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: CD47 is a broadly expressed cell surface glycoprotein associated with immune evasion. Interaction with the inhibitory receptor signal regulatory protein alpha (SIRPα), primarily expressed on myeloid cells, normally serves to restrict effector function (eg, phagocytosis and immune cell homeostasis). CD47/SIRPα antagonists, commonly referred to as 'macrophage checkpoint' inhibitors, are being developed as cancer interventions. SRF231 is an investigational fully human IgG4 anti-CD47 antibody that is currently under evaluation in a phase 1 clinical trial. The development and preclinical characterization of SRF231 are reported here. METHODS: SRF231 was characterized in assays designed to probe CD47/SIRPα blocking potential and effects on red blood cell (RBC) phagocytosis and agglutination. Additionally, SRF231-mediated phagocytosis and cell death were assessed in macrophage:tumor cell in vitro coculture systems. Further mechanistic studies were conducted within these coculture systems to ascertain the dependency of SRF231-mediated antitumor activity on Fc receptor engagement vs CD47/SIRPα blockade. In vivo, SRF231 was evaluated in a variety of hematologic xenograft models, and the mechanism of antitumor activity was assessed using cytokine and macrophage infiltration analyses following SRF231 treatment. RESULTS: SRF231 binds CD47 and disrupts the CD47/SIRPα interaction without causing hemagglutination or RBC phagocytosis. SRF231 exerts antitumor activity in vitro through both phagocytosis and cell death in a manner dependent on the activating Fc-gamma receptor (FcγR), CD32a. Through its Fc domain, SRF231 engagement with macrophage-derived CD32a serves dual purposes by eliciting FcγR-mediated phagocytosis of cancer cells and acting as a scaffold to drive CD47-mediated death signaling into tumor cells. Robust antitumor activity occurs across multiple hematologic xenograft models either as a single agent or in combination with rituximab. In tumor-bearing mice, SRF231 increases tumor macrophage infiltration and induction of the macrophage cytokines, mouse chemoattractant protein 1 and macrophage inflammatory protein 1 alpha. Macrophage depletion results in diminished SRF231 antitumor activity, underscoring a mechanistic role for macrophage engagement by SRF231. CONCLUSION: SRF231 elicits antitumor activity via apoptosis and phagocytosis involving macrophage engagement in a manner dependent on the FcγR, CD32a.
Assuntos
Antígeno CD47/metabolismo , Neoplasias/genética , Receptores de IgG/metabolismo , Animais , Humanos , Camundongos , Neoplasias/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Inhibitors of heat shock protein (Hsp) 90 induce apoptosis in multiple myeloma (MM) cells, but the molecular mechanisms underlying this cytotoxic outcome are not clear. Here, we investigate the effect of IPI-504, a novel and highly soluble inhibitor of the Hsp90 ATPase activity, on the unfolded protein response (UPR) in MM cells. The UPR is a stress response pathway triggered by sensors located at the endoplasmic reticulum (ER) membrane whose function is to reduce an excessive accumulation of misfolded protein in the ER. During normal development of B-lymphocytes to antibody-producing plasma cells, a partial UPR has been described, where IREalpha and ATF-6 are stimulated, whereas the third sensor, PERK, is not induced. METHODS: Levels of the activated forms of the three main UPR sensors ATF-6, XBP-1 and PERK/eIF-2 were monitored in two different MM cells lines and one non-MM cell lines under various experimental conditions including incubation with increasing concentration of IPI-504. Also, MM cells were incubated with IPI-504 and several apoptosis markers were monitored. RESULTS: We show here that a partial UPR is constitutively activated in plasma cell-derived MM cells and that IPI-504 can potently inhibit this pathway. IPI-504 achieves this by inactivating the transcription factors XBP1 and ATF6. In addition, IPI-504 also blocks the tunicamycin-induced phosphorylation of eIF2 by PERK. Dose-response and time course experiments reveal that IPI-504's inhibitory effect on the UPR parallels its cytotoxic and pro-apoptotic effects on MM cells. CONCLUSION: The results presented here suggest that the IPI-504-induced apoptosis might be, in part, mediated by the inhibition of the partial UPR. Other malignancies that rely on intact and efficient UPR to survive could be considered as new indications for Hsp90 inhibitors.
Assuntos
Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Dobramento de Proteína , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/fisiologia , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Western Blotting , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Relação Dose-Resposta a Droga , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/fisiologia , Genes Reporter/genética , Humanos , Luciferases/genética , Mieloma Múltiplo/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Fosforilação , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição , Transfecção , Tunicamicina/farmacologia , Proteína 1 de Ligação a X-Box , eIF-2 Quinase/genética , eIF-2 Quinase/fisiologiaRESUMO
PURPOSE: In the current study, we examine the effects of a novel proteasome inhibitor, NPI-0052 (salinosporamide A), on proteasome function and nuclear factor-kappaB activation and evaluate its ability to enhance treatment response in colon cancer xenografts when administered orally. EXPERIMENTAL DESIGN: The effects of treatment on nuclear factor-kappaB activation, cell cycle regulation, and apoptosis were determined. The pharmacodynamic effect of NPI-0052 on 20S proteasome function was assayed in vivo following oral and i.v. drug administration and compared with treatment with bortezomib. The effect of combined treatment with chemotherapy was determined in a colon cancer xenograft model. RESULTS: We found that NPI-0052 is a potent, well-tolerated proteasome inhibitor that has pharmacodynamic properties distinct from bortezomib in that it achieves significantly higher and more sustained levels of proteasome inhibition. When combined with chemotherapy, NPI-0052 increases apoptosis and shifts cells toward G2 cell cycle arrest. When added to chemotherapy in vivo [using combinations of 5-fluorouracil (5-FU), CPT-11, Avastin (bevacizumab), leucovorin, and oxaliplatin], NPI-0052 significantly improved the tumoricidal response and resulted in a 1.8-fold increased response to CPT-11, 5-FU, and leucovorin triple-drug combination (P=0.0002, t test), a 1.5-fold increased response to the oxaliplatin, 5-FU, and leucovorin triple-drug combination (P=0.013, t test), and a 2.3-fold greater response to the CPT-11, 5-FU, leucovorin, and Avastin regimen (P=0.00057). CONCLUSIONS: The high level of proteasome inhibition achieved by NPI-0052 is well tolerated and significantly improves the tumoricidal response to multidrug treatment in a colon cancer xenograft model. Further evaluation of this novel proteasome inhibitor in clinical trials is indicated.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Lactonas/farmacologia , Lactonas/uso terapêutico , Pirróis/farmacologia , Pirróis/uso terapêutico , Adenocarcinoma/metabolismo , Animais , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Bortezomib , Ciclo Celular/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Vias de Administração de Medicamentos , Sinergismo Farmacológico , Feminino , Humanos , Lactonas/administração & dosagem , Camundongos , Camundongos Nus , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Pirazinas/farmacologia , Pirróis/administração & dosagem , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
17-Allylamino-17-demethoxygeldanamycin (17-AAG)1 is a semisynthetic inhibitor of the 90 kDa heat shock protein (Hsp90) currently in clinical trials for the treatment of cancer. However, 17-AAG faces challenging formulation issues due to its poor solubility. Here we report the synthesis and evaluation of a highly soluble hydroquinone hydrochloride derivative of 17-AAG, 1a (IPI-504), and several of the physiological metabolites. These compounds show comparable binding affinity to human Hsp90 and its endoplasmic reticulum (ER) homologue, the 94 kDa glucose regulated protein (Grp94). Furthermore, the compounds inhibit the growth of the human cancer cell lines SKBR3 and SKOV3, which overexpress Hsp90 client protein Her2, and cause down-regulation of Her2 as well as induction of Hsp70 consistent with Hsp90 inhibition. There is a clear correlation between the measured binding affinity of the compounds and their cellular activities. Upon the basis of its potent activity against Hsp90 and a significant improvement in solubility, 1a is currently under evaluation in Phase I clinical trials for cancer.
Assuntos
Antineoplásicos/síntese química , Benzoquinonas/síntese química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Hidroquinonas/síntese química , Lactamas Macrocíclicas/síntese química , Rifabutina/análogos & derivados , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzoquinonas/química , Benzoquinonas/farmacologia , Ligação Competitiva , Linhagem Celular Tumoral , Cães , Ensaios de Seleção de Medicamentos Antitumorais , Polarização de Fluorescência , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP90/química , Humanos , Hidroquinonas/química , Hidroquinonas/farmacologia , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacologia , Proteínas de Membrana/química , Modelos Moleculares , Isoformas de Proteínas/química , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/biossíntese , Rifabutina/síntese química , Rifabutina/química , Rifabutina/farmacologia , Solubilidade , Relação Estrutura-Atividade , ÁguaRESUMO
Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (26 or IPI-549) with >100-fold selectivity over other lipid and protein kinases. IPI-549 demonstrates favorable pharmacokinetic properties and robust inhibition of PI3K-γ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with advanced solid tumors.
RESUMO
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.
Assuntos
Antineoplásicos/síntese química , Inibidores da Síntese de Proteínas/síntese química , Triterpenos/síntese química , Regiões 5' não Traduzidas , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Genes Reporter , Humanos , Luciferases/biossíntese , Luciferases/genética , Camundongos , Microssomos Hepáticos/metabolismo , Inibidores da Síntese de Proteínas/farmacocinética , Inibidores da Síntese de Proteínas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Triterpenos/farmacocinética , Triterpenos/farmacologiaRESUMO
IPI-504, a water-soluble ansamycin analogue currently being investigated in clinical trials, is a potent inhibitor of the protein chaperone heat shock protein 90 (Hsp90). Inhibition of Hsp90 by IPI-504 triggers the degradation of important oncogenic client proteins. In cells, the free base of IPI-504 hydroquinone exists in a dynamic redox equilibrium with its corresponding quinone (17-AAG); the hydroquinone form binding 50 times more tightly to Hsp90. It has been proposed recently that the NAD(P)H:quinone oxidoreductase NQO1 can produce the active hydroquinone and could be essential for the activity of IPI-504. Here, we have devised a method to directly measure the intracellular ratio of hydroquinone to quinone (HQ/Q) and have applied this measurement to correlate NQO1 enzyme abundance with HQ/Q ratio and cellular activity of IPI-504 in 30 cancer cell lines. Interestingly, the intracellular HQ/Q ratio was correlated with NQO1 levels only in a subset of cell lines and overall was poorly correlated with the growth inhibitory activity of IPI-504. Although artificial overexpression of NQO1 is able to increase the level of hydroquinone and cell sensitivity to IPI-504, it has little effect on the activity of 17-amino-17-demethoxy-geldanamycin, the major active metabolite of IPI-504. This finding could provide an explanation for the biological activity of IPI-504 in xenograft models of cell lines that are not sensitive to IPI-504 in vitro. Our results suggest that NQO1 activity is not a determinant of IPI-504 activity in vivo and, therefore, unlikely to become an important resistance mechanism to IPI-504 in the clinic.
Assuntos
Benzoquinonas/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Animais , Benzoquinonas/metabolismo , Linhagem Celular Tumoral , Células HCT116 , Proteínas de Choque Térmico HSP90/metabolismo , Células HT29 , Humanos , Hidroquinonas/metabolismo , Immunoblotting , Células K562 , Lactamas Macrocíclicas/metabolismo , Masculino , Camundongos , Camundongos Nus , Mutação , NAD(P)H Desidrogenase (Quinona)/genética , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent evidence suggests that blocking aberrant hedgehog pathway signaling may be a promising therapeutic strategy for the treatment of several types of cancer. Cyclopamine, a plant Veratrum alkaloid, is a natural product antagonist of the hedgehog pathway. In a previous report, a seven-membered D-ring semisynthetic analogue of cyclopamine, IPI-269609 (2), was shown to have greater acid stability and better aqueous solubility compared to cyclopamine. Further modifications of the A-ring system generated three series of analogues with improved potency and/or solubility. Lead compounds from each series were characterized in vitro and evaluated in vivo for biological activity and pharmacokinetic properties. These studies led to the discovery of IPI-926 (compound 28), a novel semisynthetic cyclopamine analogue with substantially improved pharmaceutical properties and potency and a favorable pharmacokinetic profile relative to cyclopamine and compound 2. As a result, complete tumor regression was observed in a Hh-dependent medulloblastoma allograft model after daily oral administration of 40 mg/kg of compound 28.
Assuntos
Descoberta de Drogas , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Transdução de Sinais/efeitos dos fármacos , Alcaloides de Veratrum/administração & dosagem , Alcaloides de Veratrum/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular , Humanos , Fígado/citologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Estereoisomerismo , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacocinéticaRESUMO
Salinosporamide A ( 1 (NPI-0052)) is a potent, monochlorinated 20S proteasome inhibitor in clinical trials for the treatment of cancer. To elucidate the role of the chlorine leaving group (LG), we synthesized analogues with a range of LG potentials and determined their IC 50 values for inhibition of chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L) activities of 20S proteasomes. Proteasome activity was also determined before and after attempted removal of the inhibitors by dialysis. Analogues bearing substituents with good LG potential exhibited the greatest potency and prolonged duration of proteasome inhibition, with no recovery after 24 h of dialysis. In contrast, activity was restored after =12 h in the case of non-LG analogues. Intermediate results were observed for fluorosalinosporamide, with poor LG potential. Kinetic studies indicate that 1 acts as a classical slow, tight inhibitor of the CT-L, T-L, and C-L activities and that inhibition occurs via a two-step mechanism involving reversible recognition followed by rate-limiting formation of a covalent enzyme-inhibitor complex.
Assuntos
Lactamas/síntese química , Lactamas/farmacologia , Lactonas/síntese química , Lactonas/farmacologia , Inibidores de Proteassoma , Pirróis/síntese química , Pirróis/farmacologia , Animais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidrólise , Cinética , Lactamas/química , Lactonas/química , Modelos Moleculares , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Pirróis/química , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Herein is reported the synthesis of a novel class of hedgehog antagonists derived from cyclopamine. The acid sensitive D-ring of cyclopamine was homologated utilizing a sequence of chemoselective cyclopropanation and stereoselective acid-catalyzed rearrangement. Further modification of the A/B-ring homoallylic alcohol to the conjugated ketone led to the discovery of new cyclopamine analogues with improved pharmaceutical properties and in vitro potency (EC 50) ranging from 10 to 1000 nM.
Assuntos
Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Transdução de Sinais/efeitos dos fármacos , Alcaloides de Veratrum/síntese química , Administração Oral , Estrutura Molecular , Relação Estrutura-Atividade , Alcaloides de Veratrum/administração & dosagem , Alcaloides de Veratrum/químicaRESUMO
Heat shock protein 90 (Hsp90) is an emerging therapeutic target of interest for the treatment of cancer. Its role in protein homeostasis and the selective chaperoning of key signaling proteins in cancer survival and proliferation pathways has made it an attractive target of small molecule therapeutic intervention. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), the most studied agent directed against Hsp90, suffers from poor physical-chemical properties that limit its clinical potential. Therefore, there exists a need for novel, patient-friendly Hsp90-directed agents for clinical investigation. IPI-504, the highly soluble hydroquinone hydrochloride derivative of 17-AAG, was synthesized as an Hsp90 inhibitor with favorable pharmaceutical properties. Its biochemical and biological activity was profiled in an Hsp90-binding assay, as well as in cancer-cell assays. Furthermore, the metabolic profile of IPI-504 was compared with that of 17-AAG, a geldanamycin analog currently in clinical trials. The anti-tumor activity of IPI-504 was tested as both a single agent as well as in combination with bortezomib in myeloma cell lines and in vivo xenograft models, and the retention of IPI-504 in tumor tissue was determined. In conclusion, IPI-504, a potent inhibitor of Hsp90, is efficacious in cellular and animal models of myeloma. It is synergistically efficacious with the proteasome inhibitor bortezomib and is preferentially retained in tumor tissues relative to plasma. Importantly, it was observed that IPI-504 interconverts with the known agent 17-AAG in vitro and in vivo via an oxidation-reduction equilibrium, and we demonstrate that IPI-504 is the slightly more potent inhibitor of Hsp90.