RESUMEN
The propensity of naive CD4 T cells to become T helper (Th) type 2 cells correlates with susceptibility to infection by the protozoal parasite Leishmania major. Using genetic linkage analysis, we earlier identified Dice1 as a Th2 cell bias-controlling quantitative trait locus on chromosome 16. Using interval-specific congenic mapping, we now resolve Dice1 into two independent genetic loci, Dice1.1 and Dice1.2, which control Il4 expression from naive Th cells and thereby indirectly control Th2 cell bias. Interestingly, only one of the two congenic intervals containing Dice1.1 and Dice1.2, respectively, also contained an L. major response locus, indicating that L. major responsiveness can be insensitive to determinants that influence Th2 cell bias by controlling naive T cell Il4 expression. These results lay the groundwork for identifying the Dice1.1 and Dice1.2 genes controlling naive T cell Il4 expression and L. major responses, and for testing whether these control other Th2 cell-dependent processes such as worm expulsion, allergic asthma, and dermatitis.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Activación de Linfocitos/genética , Sitios de Carácter Cuantitativo/genética , Células Th2/inmunología , Animales , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Interleucina-4/biosíntesis , Interleucina-4/genética , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/patología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Sitios de Carácter Cuantitativo/inmunologíaRESUMEN
Immunotherapy for breast cancer using cytotoxic T cells (CTL) is hindered by the lack of well-characterized breast cancer antigens that are expressed in most breast tumor cells and recognized by CD8+ CTL. A recently described breast tissue differentiation antigen, NY-BR-1, is expressed in >80% breast tumors and elicits a humoral response in a subset of breast cancer patients. To identify potential NY-BR-1 epitopes that are recognized by CTL, CD8+ T cells were stimulated in vitro with autologous dendritic cells pulsed with NY-BR-1 peptides that were predicted to bind to HLA-A2. In multiple normal female donors and breast cancer patients, specific CD8+ CTL responses were detected by enzyme-linked immunospot assay against several NY-BR-1 peptides after two cycles of stimulation. CD8+ CTL clones against three NY-BR-1 epitopes were isolated and recognized peptide-pulsed target cells with high avidity. T-cell clones specific for one of the NY-BR-1 epitopes (p904) also recognized breast tumor cells expressing NY-BR-1, NY-BR-1(-) cells transfected with a cDNA encoding the NY-BR-1 protein, and autologous dendritic cells pulsed with opsonized NY-BR-1+ breast tumor cells. Taken together, these results show that the p904 epitope derived from NY-BR-1 is efficiently processed and presented endogenously and identify NY-BR-1 as a promising target for T-cell-based immunotherapy for breast cancer.
Asunto(s)
Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/inmunología , Epítopos de Linfocito T/inmunología , Linfocitos T Citotóxicos/inmunología , Presentación de Antígeno , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Células Clonales , Células Dendríticas/inmunología , Femenino , Antígenos HLA-A/inmunología , Antígeno HLA-A2 , Humanos , Fragmentos de Péptidos/inmunología , TransfecciónRESUMEN
p21-activated kinase 1 (PAK1) has an important role in transducing signals in several oncogenic pathways. The concept of inhibiting this kinase has garnered significant interest over the past decade, particularly for targeting cancers associated with PAK1 amplification. Animal studies with the selective group I PAK (pan-PAK1, 2, 3) inhibitor G-5555 from the pyrido[2,3-d]pyrimidin-7-one class uncovered acute toxicity with a narrow therapeutic window. To attempt mitigating the toxicity, we introduced significant structural changes, culminating in the discovery of the potent pyridone side chain analogue G-9791. Mouse tolerability studies with this compound, other members of this series, and compounds from two structurally distinct classes revealed persistent toxicity and a correlation of minimum toxic concentrations and PAK1/2 mediated cellular potencies. Broad screening of selected PAK inhibitors revealed PAK1, 2, and 3 as the only overlapping targets. Our data suggest acute cardiovascular toxicity resulting from the inhibition of PAK2, which may be enhanced by PAK1 inhibition, and cautions against continued pursuit of pan-group I PAK inhibitors in drug discovery.
Asunto(s)
Enfermedades Cardiovasculares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Enfermedad Aguda , Animales , Relación Dosis-Respuesta a Droga , Femenino , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/química , Piridonas , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Quinasas p21 Activadas/metabolismoRESUMEN
Signaling pathways intersecting with the p21-activated kinases (PAKs) play important roles in tumorigenesis and cancer progression. By recognizing that the limitations of FRAX1036 (1) were chiefly associated with the highly basic amine it contained, we devised a mitigation strategy to address several issues such as hERG activity. The 5-amino-1,3-dioxanyl moiety was identified as an effective means of reducing pK a and logP simultaneously. When positioned properly within the scaffold, this group conferred several benefits including potency, pharmacokinetics, and selectivity. Mouse xenograft PK/PD studies were carried out using an advanced compound, G-5555 (12), derived from this approach. These studies concluded that dose-dependent pathway modulation was achievable and paves the way for further in vivo investigations of PAK1 function in cancer and other diseases.
RESUMEN
Checkpoint kinase 1 (ChK1) plays a key role in the DNA damage response, facilitating cell-cycle arrest to provide sufficient time for lesion repair. This leads to the hypothesis that inhibition of ChK1 might enhance the effectiveness of DNA-damaging therapies in the treatment of cancer. Lead compound 1 (GNE-783), the prototype of the 1,7-diazacarbazole class of ChK1 inhibitors, was found to be a highly potent inhibitor of acetylcholine esterase (AChE) and unsuitable for development. A campaign of analogue synthesis established SAR delineating ChK1 and AChE activities and allowing identification of new leads with improved profiles. In silico docking using a model of AChE permitted rationalization of the observed SAR. Compounds 19 (GNE-900) and 30 (GNE-145) were identified as selective, orally bioavailable ChK1 inhibitors offering excellent in vitro potency with significantly reduced AChE activity. In combination with gemcitabine, these compounds demonstrate an in vivo pharmacodynamic effect and are efficacious in a mouse p53 mutant xenograft model.
Asunto(s)
Acetilcolinesterasa/metabolismo , Carbazoles/química , Carbazoles/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Acetilcolinesterasa/química , Acetilcolinesterasa/farmacocinética , Acetilcolinesterasa/uso terapéutico , Animales , Compuestos Aza/química , Compuestos Aza/farmacocinética , Compuestos Aza/farmacología , Compuestos Aza/uso terapéutico , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacocinética , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/uso terapéutico , Cristalografía por Rayos X , Perros , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/química , RatasRESUMEN
Checkpoint kinase 1 (ChK1) is a serine/threonine kinase that functions as a central mediator of the intra-S and G2-M cell-cycle checkpoints. Following DNA damage or replication stress, ChK1-mediated phosphorylation of downstream effectors delays cell-cycle progression so that the damaged genome can be repaired. As a therapeutic strategy, inhibition of ChK1 should potentiate the antitumor effect of chemotherapeutic agents by inactivating the postreplication checkpoint, causing premature entry into mitosis with damaged DNA resulting in mitotic catastrophe. Here, we describe the characterization of GNE-900, an ATP-competitive, selective, and orally bioavailable ChK1 inhibitor. In combination with chemotherapeutic agents, GNE-900 sustains ATR/ATM signaling, enhances DNA damage, and induces apoptotic cell death. The kinetics of checkpoint abrogation seems to be more rapid in p53-mutant cells, resulting in premature mitotic entry and/or accelerated cell death. Importantly, we show that GNE-900 has little single-agent activity in the absence of chemotherapy and does not grossly potentiate the cytotoxicity of gemcitabine in normal bone marrow cells. In vivo scheduling studies show that optimal administration of the ChK1 inhibitor requires a defined lag between gemcitabine and GNE-900 administration. On the refined combination treatment schedule, gemcitabine's antitumor activity against chemotolerant xenografts is significantly enhanced and dose-dependent exacerbation of DNA damage correlates with extent of tumor growth inhibition. In summary, we show that in vivo potentiation of gemcitabine activity is mechanism based, with optimal efficacy observed when S-phase arrest and release is followed by checkpoint abrogation with a ChK1 inhibitor.
Asunto(s)
Desoxicitidina/análogos & derivados , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Quinasas/metabolismo , Piridinas/administración & dosificación , Pirroles/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Desoxicitidina/administración & dosificación , Humanos , Mitosis/efectos de los fármacos , Mitosis/genética , Neoplasias/genética , Neoplasias/patología , Fosforilación/efectos de los fármacos , GemcitabinaRESUMEN
We have recently reported a series of tetrahydroquinazoline (THQ) mTOR inhibitors that produced a clinical candidate 1 (GDC-0349). Through insightful design, we hoped to discover and synthesize a new series of small molecule inhibitors that could attenuate CYP3A4 time-dependent inhibition commonly observed with the THQ scaffold, maintain or improve aqueous solubility and oral absorption, reduce free drug clearance, and selectively increase mTOR potency. Through key in vitro and in vivo studies, we demonstrate that a pyrimidoaminotropane based core was able to address each of these goals. This effort culminated in the discovery of 20 (GNE-555), a highly potent, selective, metabolically stable, and efficacious mTOR inhibitor.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Tropanos/farmacología , Cromatografía Liquida , Inhibidores Enzimáticos/química , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Tropanos/químicaRESUMEN
Aberrant activation of the PI3K-Akt-mTOR signaling pathway has been observed in human tumors and tumor cell lines, indicating that these protein kinases may be attractive therapeutic targets for treating cancer. Optimization of advanced lead 1 culminated in the discovery of clinical development candidate 8h, GDC-0349, a potent and selective ATP-competitive inhibitor of mTOR. GDC-0349 demonstrates pathway modulation and dose-dependent efficacy in mouse xenograft cancer models.
RESUMEN
Selective inhibitors of mammalian target of rapamycin (mTOR) kinase based upon saturated heterocycles fused to a pyrimidine core were designed and synthesized. Each series produced compounds with K(i) < 10 nM for the mTOR kinase and >500-fold selectivity over closely related PI3 kinases. This potency translated into strong pathway inhibition, as measured by phosphorylation of mTOR substrate proteins and antiproliferative activity in cell lines with a constitutively active PI3K pathway. Two compounds exhibiting suitable mouse PK were profiled in in vivo tumor models and were shown to suppress mTORC1 and mTORC2 signaling for over 12 h when dosed orally. Both compounds were additionally shown to suppress tumor growth in vivo in a PC3 prostate cancer model over a 14 day study.
Asunto(s)
Antineoplásicos/síntesis química , Complejos Multiproteicos/antagonistas & inhibidores , Pirimidinas/síntesis química , Pirroles/síntesis química , Quinazolinas/síntesis química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Disponibilidad Biológica , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Desnudos , Simulación del Acoplamiento Molecular , Trasplante de Neoplasias , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Neoplasias de la Próstata , Pirimidinas/química , Pirimidinas/farmacología , Pirroles/química , Pirroles/farmacología , Quinazolinas/química , Quinazolinas/farmacología , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
A series of inhibitors of mTOR kinase based on a quaternary-substituted dihydrofuropyrimidine was designed and synthesized. The most potent compounds in this series inhibited mTOR kinase with K(i) < 1.0 nM and were highly (>100×) selective for mTOR over the closely related PI3 kinases. Compounds in this series showed inhibition of the pathway and antiproliferative activity in cell-based assays. Furthermore, these compounds had excellent mouse PK, and showed a robust PK-PD relationship in a mouse model of cancer.