RESUMEN
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.
Asunto(s)
Antineoplásicos , Proteína-Arginina N-Metiltransferasas , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , Humanos , Animales , Ratones , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Antineoplásicos/síntesis química , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias/tratamiento farmacológico , Encéfalo/metabolismo , Relación Estructura-ActividadRESUMEN
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.
RESUMEN
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.
Asunto(s)
Benzoquinonas/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Taxoides/administración & dosificación , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Benzoquinonas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Docetaxel , Regulación hacia Abajo , Sinergismo Farmacológico , Humanos , Lactamas Macrocíclicas/farmacología , Neoplasias Pulmonares/metabolismo , Masculino , Taxoides/farmacología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Artritis/tratamiento farmacológico , Asma/tratamiento farmacológico , Modelos Animales de Enfermedad , Isoquinolinas/farmacología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Purinas/farmacología , Animales , Artritis/inducido químicamente , Artritis/inmunología , Asma/inducido químicamente , Asma/inmunología , Colágeno Tipo II , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Isoquinolinas/química , Lupus Eritematoso Sistémico/inmunología , Estructura Molecular , Ovalbúmina , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/metabolismo , Purinas/química , Ratas , Ratas Endogámicas Lew , Ratas Wistar , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antineoplásicos/síntesis química , Inhibidores de la Síntesis de la Proteína/síntesis química , Triterpenos/síntesis química , Regiones no Traducidas 5' , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Genes Reporteros , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Microsomas Hepáticos/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacocinética , Inhibidores de la Síntesis de la Proteína/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Triterpenos/farmacocinética , Triterpenos/farmacologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacocinética , Benzoquinonas/farmacocinética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacocinética , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Antineoplásicos/farmacología , Benzoquinonas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Lactamas Macrocíclicas/farmacología , Neoplasias Pulmonares/metabolismo , Ratones , Ratones DesnudosRESUMEN
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.
Asunto(s)
Lactamas/síntesis química , Lactamas/farmacología , Lactonas/síntesis química , Lactonas/farmacología , Inhibidores de Proteasoma , Pirroles/síntesis química , Pirroles/farmacología , Animales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hidrólisis , Cinética , Lactamas/química , Lactonas/química , Modelos Moleculares , Estructura Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Pirroles/química , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Benzoquinonas/química , Benzoquinonas/farmacología , Proteínas HSP90 de Choque Térmico/metabolismo , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacología , Neoplasias/metabolismo , Animales , Antineoplásicos/farmacocinética , Benzoquinonas/farmacocinética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Lactamas Macrocíclicas/farmacocinética , Ratones , Ratones Endogámicos BALB C , Microsomas Hepáticos/efectos de los fármacos , Neoplasias/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Antineoplásicos/síntesis química , Benzoquinonas/síntesis química , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Hidroquinonas/síntesis química , Lactamas Macrocíclicas/síntesis química , Rifabutina/análogos & derivados , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Benzoquinonas/química , Benzoquinonas/farmacología , Unión Competitiva , Línea Celular Tumoral , Perros , Ensayos de Selección de Medicamentos Antitumorales , Polarización de Fluorescencia , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/química , Humanos , Hidroquinonas/química , Hidroquinonas/farmacología , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacología , Proteínas de la Membrana/química , Modelos Moleculares , Isoformas de Proteínas/química , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/biosíntesis , Rifabutina/síntesis química , Rifabutina/química , Rifabutina/farmacología , Solubilidad , Relación Estructura-Actividad , AguaRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , ADN Bacteriano/metabolismo , Secuencia de Bases , Cartilla de ADN , Dimerización , Proteínas de Escherichia coli , CinéticaRESUMEN
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.