RESUMO
It has been shown that PRMT5 inhibition by small molecules can selectively kill cancer cells with homozygous deletion of the MTAP gene if the inhibitors can leverage the consequence of MTAP deletion, namely, accumulation of the MTAP substrate MTA. Herein, we describe the discovery of TNG908, a potent inhibitor that binds the PRMT5·MTA complex, leading to 15-fold-selective killing of MTAP-deleted (MTAP-null) cells compared to MTAPintact (MTAP WT) cells. TNG908 shows selective antitumor activity when dosed orally in mouse xenograft models, and its physicochemical properties are amenable for crossing the blood-brain barrier (BBB), supporting clinical study for the treatment of both CNS and non-CNS tumors with MTAP loss.
Assuntos
Antineoplásicos , Proteína-Arginina N-Metiltransferases , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Humanos , Animais , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Antineoplásicos/síntese química , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/tratamento farmacológico , Encéfalo/metabolismo , Relação Estrutura-AtividadeRESUMO
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
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
The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding "step-size", m=3.9(+/-1.3)bp step(-1), with an average unwinding rate of k(U)=196(+/-77)steps s(-1) (mk(U)=790(+/-23)bps(-1)) at 25.0 degrees C (10mM MgCl(2), 30 mM NaCl (pH 7.0), 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding.
Assuntos
DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleases/metabolismo , Biologia Molecular/métodos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Bases , DNA Helicases/química , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Exodesoxirribonuclease V , Exodesoxirribonucleases/química , Cinética , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes , Recombinação 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
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
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.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Benzoquinonas/química , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacologia , Neoplasias/metabolismo , Animais , Antineoplásicos/farmacocinética , Benzoquinonas/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Lactamas Macrocíclicas/farmacocinética , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/efeitos dos fármacos , Neoplasias/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Escherichia coli UvrD protein is a 3' to 5' SF1 helicase required for DNA repair as well as DNA replication of certain plasmids. We have shown previously that UvrD can self-associate to form dimers and tetramers in the absence of DNA, but that a UvrD dimer is required to form an active helicase-DNA complex in vitro. Here we have used pre-steady state, chemical quenched flow methods to examine the kinetic mechanism for formation of the active, dimeric helicase-DNA complex. Experiments were designed to examine the steps leading to formation of the active complex, separate from the subsequent DNA unwinding steps. The results show that the active dimeric complex can form via two pathways. The first, faster path involves direct binding to the DNA substrate of a pre-assembled UvrD dimer (dimer path), whereas the second, slower path proceeds via sequential binding to the DNA substrate of two UvrD monomers (monomer path), which then assemble on the DNA to form the dimeric helicase. The rate-limiting step within the monomer pathway involves dimer assembly on the DNA. These results show that UvrD dimers that pre-assemble in the absence of DNA are intermediates along the pathway to formation of the functional dimeric UvrD helicase.