RESUMEN
The cell cycle is an evolutionarily conserved process necessary for mammalian cell growth and development. Because cell-cycle aberrations are a hallmark of cancer, this process has been the target of anti-cancer therapeutics for decades. However, despite numerous clinical trials, cell-cycle-targeting agents have generally failed in the clinic. This review briefly examines past cell-cycle-targeted therapeutics and outlines how experience with these agents has provided valuable insight to refine and improve anti-mitotic strategies. An overview of emerging anti-mitotic approaches with promising pre-clinical results is provided, and the concept of exploiting the genomic instability of tumor cells through therapeutic inhibition of mitotic checkpoints is discussed. We believe this strategy has a high likelihood of success given its potential to enhance therapeutic index by targeting tumor-specific vulnerabilities. This reasoning stimulated our development of novel inhibitors targeting the critical regulators of genomic stability and the mitotic checkpoint: AURKA, PLK4, and Mps1/TTK.
Asunto(s)
Antineoplásicos/farmacología , Mitosis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Aurora Quinasa A/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inestabilidad Genómica/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Neoplasias/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidoresRESUMEN
Loss of cell-cycle control is a hallmark of human cancer. Cell-cycle checkpoints are essential for maintaining genome integrity and balanced growth and division. They are specifically deregulated in cancer cells and contain regulators that represent potential therapeutic targets. Monopolar spindle 1 (Mps1; also known as TTK protein kinase) is a core component of the spindle assembly checkpoint (SAC), a genome-surveillance mechanism that is important for cell survival, and has emerged as a candidate target for anticancer therapy. Here, we report the cellular and antitumor effects of CFI-402257, a potent (Mps1 Ki = 0.09 ± 0.02 nM; cellular Mps1 EC50 = 6.5 ± 0.5 nM), highly selective, and orally active small-molecule inhibitor of Mps1 that was identified through a drug-discovery program. Human cancer cells treated with CFI-402257 exhibit effects consistent with Mps1 kinase inhibition, specifically SAC inactivation, leading to chromosome missegregation, aneuploidy, and ultimately cell death. Oral administration of CFI-402257 in monotherapy or in combination with an anti-programmed cell death 1 (PD-1) antibody in mouse models of human cancer results in inhibition of tumor growth at doses that are well-tolerated. Our findings provide a rationale for the clinical evaluation of CFI-402257 in patients with solid tumors.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Pirazoles/administración & dosificación , Pirazoles/farmacocinética , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética , Interferencia de ARN , ARN Interferente Pequeño/genética , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
TTK/Mps1 is a key kinase controlling progression of cell division via participation in the mitotic spindle assembly checkpoint and is overexpressed in a number of human cancers. Herein we report the discovery of 4-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)benzamides as a potent, novel class of TTK inhibitors. The series was identified by means of bioisosteric replacement of the related imidazopyrazine and imidazopyridazine scaffolds. Optimization led to the identification of compounds with excellent potency (Ki=0.8nM) and exceptional kinase selectivity. The SAR indicates a strong dependence of activity on the presence of the N-cyclopropyl-2-methylbenzamide moiety delineating the geometry for 1½ type kinase inhibitor. Molecular modeling indicates the extensive and optimal contacts, mediated through H-bonds and hydrophobic interactions, are responsible for the selectivity and potency of the inhibitors. The compounds demonstrate a strong anti-proliferative activity in a panel of human cancer cell lines (HCT116 GI50<15nM) and good rodent pharmacokinetics (oral %F 97%).
Asunto(s)
Antineoplásicos/farmacología , Benzamidas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Triazinas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Benzamidas/administración & dosificación , Benzamidas/química , Disponibilidad Biológica , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Ratas , Relación Estructura-Actividad , Triazinas/administración & dosificación , Triazinas/químicaRESUMEN
Previous efforts from our laboratory demonstrated that (E)-3-((3-(E)-vinylaryl)-1H-indazol-6-yl)methylene)-indolin-2-ones are potent PLK4 inhibitors with in vivo anticancer efficacy upon IP dosing. As part of a continued effort to develop selective and orally efficacious inhibitors, we examined variations on this theme wherein 'directly-linked' aromatics, pendant from the indazole core, replace the arylvinyl moiety. Herein, we describe the design and optimization of this series which was ultimately superseded by (3-aryl-1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones. The latter compounds are potent and selective inhibitors of PLK4 with oral exposure in rodents and in vivo anticancer activity. Compound 13b, in particular, has a bioavailability of 22% and achieved a 96% tumor growth inhibition in an MDA-MB-468 xenograft study.
Asunto(s)
Antineoplásicos/farmacología , Indoles/química , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Área Bajo la Curva , Línea Celular Tumoral , Diseño de Fármacos , Xenoinjertos , Humanos , Indoles/administración & dosificación , Indoles/farmacocinética , Ratones , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , RatasRESUMEN
TTK kinase was identified by in-house siRNA screen and pursued as a tractable, novel target for cancer treatment. A screening campaign and systematic optimization, supported by computer modeling led to an indazole core with key sulfamoylphenyl and acetamido moieties at positions 3 and 5, respectively, establishing a novel chemical class culminating in identification of 72 (CFI-400936). This potent inhibitor of TTK (IC50=3.6nM) demonstrated good activity in cell based assay and selectivity against a panel of human kinases. A co-complex TTK X-ray crystal structure and results of a xenograft study with TTK inhibitors from this class are described.
Asunto(s)
Amidas/farmacología , Bencenoacetamidas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Descubrimiento de Drogas , Indazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Amidas/síntesis química , Amidas/química , Bencenoacetamidas/síntesis química , Bencenoacetamidas/química , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Indazoles/síntesis química , Indazoles/química , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
CFI-400945 is a selective oral polo-like kinase 4 (PLK4) inhibitor that regulates centriole duplication. PLK4 is aberrantly expressed in patients with acute myeloid leukemia (AML). Preclinical studies indicate that CFI-400945 has potent in vivo efficacy in hematological malignancies and xenograft models, with activity in cells harboring TP53 mutations. In this phase 1 study in very high-risk patients with relapsed/refractory AML and myelodysplastic syndrome (MDS) (NCT03187288), 13 patients were treated with CFI-400945 continuously in dose escalation from 64 mg/day to 128 mg/day. Three of the 9 efficacy evaluable AML patients achieved complete remission (CR). Two of 4 AML patients (50%) with TP53 mutations and complex monosomal karyotype achieved a CR with 1 patient proceeding to allogenic stem cell transplant. A third patient with TP53 mutated AML had a significant reduction in marrow blasts by > 50% with an improvement in neutrophil and platelet counts. Responses were observed after 1 cycle of therapy. Dose-limiting toxicity was enteritis/colitis. A monotherapy and combination therapy study with a newer crystal form of CFI-400945 in patients with AML, MDS and chronic myelomonocytic leukemia (CMML) is ongoing (NCT04730258).
Asunto(s)
Indazoles , Indoles , Leucemia Mieloide Aguda , Leucemia Mielomonocítica Crónica , Síndromes Mielodisplásicos , Humanos , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/genética , Leucemia Mielomonocítica Crónica/tratamiento farmacológico , Supervivencia sin Enfermedad , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Receptor tyrosine kinases (RTKs) control a wide variety of processes in multicellular organisms, including proliferation, differentiation, migration and survival. Their activity is tightly controlled through the coordinated action of both positive and negative regulators that function at multiple levels of the signal transduction cascade, and at different time points within the growth-factor-induced response. When this process goes awry, the outcome can be developmental defects and malignancy. Sprouty (Spry) proteins represent a major class of ligand-inducible inhibitors of RTK-dependent signaling pathways. New biochemical and genetic evidence indicates specific roles of the Spry genes in development and multiple modes of action of the Spry proteins in regulation of the RTK-induced response.
Asunto(s)
Retroalimentación Fisiológica , Proteínas de la Membrana/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Transducción de Señal , Secuencia de Aminoácidos , Animales , Transformación Celular Neoplásica , Factor de Crecimiento Epidérmico/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes Supresores de Tumor , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidoresRESUMEN
Sprouty proteins are recently identified receptor tyrosine kinase (RTK) inhibitors potentially involved in many developmental processes. Here, we report that Sprouty proteins become tyrosine phosphorylated after growth factor treatment. We identified Tyr55 as a key residue for Sprouty2 phosphorylation and showed that phosphorylation was required for Sprouty2 to inhibit RTK signaling, because a mutant Sprouty2 lacking Tyr55 augmented signaling. We found that tyrosine phosphorylation of Sprouty2 affected neither its subcellular localization nor its interaction with Grb2, FRS2/SNT, or other Sprouty proteins. In contrast, Sprouty2 tyrosine phosphorylation was necessary for its binding to the Src homology 2-like domain of c-Cbl after fibroblast growth factor (FGF) stimulation. To determine whether c-Cbl was required for Sprouty2-dependent cellular events, Sprouty2 was introduced into c-Cbl-wild-type and -null fibroblasts. Sprouty2 efficiently inhibited FGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 in c-Cbl-null fibroblasts, thus indicating that the FGF-dependent binding of c-Cbl to Sprouty2 was dispensable for its inhibitory activity. However, c-Cbl mediates polyubiquitylation/proteasomal degradation of Sprouty2 in response to FGF. Last, using Src-family pharmacological inhibitors and dominant-negative Src, we showed that a Src-like kinase was required for tyrosine phosphorylation of Sprouty2 by growth factors. Thus, these data highlight a novel negative and positive regulatory loop that allows for the controlled, homeostatic inhibition of RTK signaling.
Asunto(s)
Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Proteínas/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/fisiología , Proteína Adaptadora GRB2 , Péptidos y Proteínas de Señalización Intracelular , Espacio Intracelular/ultraestructura , Proteínas de la Membrana/metabolismo , Ratones , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Células 3T3 NIH , Fosfoproteínas/metabolismo , Fosforilación , Mutación Puntual , Unión Proteica , Proteínas Serina-Treonina Quinasas , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Proteínas Tirosina Quinasas Receptoras/metabolismo , Tirosina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
This work describes a scaffold hopping exercise that begins with known imidazo[1,2-a]pyrazines, briefly explores pyrazolo[1,5-a][1,3,5]triazines, and ultimately yields pyrazolo[1,5-a]pyrimidines as a novel class of potent TTK inhibitors. An X-ray structure of a representative compound is consistent with 1(1)/2 type inhibition and provides structural insight to aid subsequent optimization of in vitro activity and physicochemical and pharmacokinetic properties. Incorporation of polar moieties in the hydrophobic and solvent accessible regions modulates physicochemical properties while maintaining potency. Compounds with enhanced oral exposure were identified for xenograft studies. The work culminates in the identification of a potent (TTK K i = 0.1 nM), highly selective, orally bioavailable anticancer agent (CFI-402257) for IND enabling studies.
RESUMEN
Previous publications from our laboratory have introduced novel inhibitors of Polo-like kinase 4 (PLK4), a mitotic kinase identified as a potential target for cancer therapy. The search for potent and selective PLK4 inhibitors yielded (E)-3-((1Hindazol-6-yl)methylene)indolin-2-ones, which were superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones, e.g., 3. The later scaffold confers improved drug-like properties and incorporates two stereogenic centers. This work reports the discovery of a novel one-pot double SN2 displacement reaction for the stereoselective installation of the desired asymmetric centers and confirms the stereochemistry of the most potent stereoisomer, e.g., 44. Subsequent work keys on the optimization of the oral exposure of nanomolar PLK4 inhibitors with potent cancer cell growth inhibitory activity. A short list of compounds with superior potency and pharmacokinetic properties in rodents and dogs was studied in mouse models of tumor growth. We conclude with the identification of compound 48 (designated CFI-400945) as a novel clinical candidate for cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Indazoles/farmacología , Indoles/farmacología , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/análisis , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Femenino , Células HCT116 , Humanos , Indazoles/química , Indazoles/farmacocinética , Indoles/química , Indoles/farmacocinética , Células MCF-7 , Masculino , Ratones Desnudos , Ratones SCID , Modelos Químicos , Estructura Molecular , Neoplasias/metabolismo , Neoplasias/patología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Polo-like kinase 4 (PLK4), a unique member of the polo-like kinase family of serine-threonine kinases, is a master regulator of centriole duplication that is important for maintaining genome integrity. Overexpression of PLK4 is found in several human cancers and is linked with a predisposition to tumorigenesis. Previous efforts to identify potent and efficacious PLK4 inhibitors resulted in the discovery of (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones, which are superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones reported herein. Optimization of this new cyclopropane-linked series was based on a computational model of a PLK4 X-ray structure and SAR attained from the analogous alkenelinked series. The racemic cyclopropane-linked compounds showed PLK4 affinity and antiproliferative activity comparable to their alkene-linked congeners with improved hysicochemical, ADME, and pharmacokinetic properties. Positive xenograft results from the MDA-MB-468 human breast cancer xenograft model for compound 18 support the investigation of PLK4 inhibitors as anticancer therapeutics. A PLK4 X-ray co-structure with racemate 18 revealed preferential binding of the 1R,2S enantiomer to the PLK4 kinase domain.
Asunto(s)
Antineoplásicos/farmacología , Indoles/farmacología , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Humanos , Indoles/química , Indoles/farmacocinética , Células MCF-7 , Ratones , Modelos Químicos , Estructura Molecular , Neoplasias/metabolismo , Neoplasias/patología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Compuestos de Espiro/química , Compuestos de Espiro/farmacocinética , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Controversy over the role of antioxidants in cancer has persisted for decades. Here, we demonstrate that synthesis of the antioxidant glutathione (GSH), driven by GCLM, is required for cancer initiation. Genetic loss of Gclm prevents a tumor's ability to drive malignant transformation. Intriguingly, these findings can be replicated using an inhibitor of GSH synthesis, but only if delivered prior to cancer onset, suggesting that at later stages of tumor progression GSH becomes dispensable potentially due to compensation from alternative antioxidant pathways. Remarkably, combined inhibition of GSH and thioredoxin antioxidant pathways leads to a synergistic cancer cell death in vitro and in vivo, demonstrating the importance of these two antioxidants to tumor progression and as potential targets for therapeutic intervention.
Asunto(s)
Antioxidantes/metabolismo , Neoplasias de la Mama/genética , Glutamato-Cisteína Ligasa/genética , Neoplasias Mamarias Animales/genética , Animales , Neoplasias de la Mama/patología , Carcinogénesis , Femenino , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/genética , Humanos , Neoplasias Mamarias Animales/tratamiento farmacológico , Neoplasias Mamarias Animales/patología , Ratones , Ratones Transgénicos , Tiorredoxinas/metabolismoRESUMEN
The acetamido and carboxamido substituted 3-(1H-indazol-3-yl)benzenesulfonamides are potent TTK inhibitors. However, they display modest ability to attenuate cancer cell growth; their physicochemical properties, and attendant pharmacokinetic parameters, are not drug-like. By eliminating the polar 3-sulfonamide group and grafting a heterocycle at the 4 position of the phenyl ring, potent inhibitors with oral exposure were obtained. An X-ray cocrystal structure and a refined binding model allowed for a structure guided approach. Systematic optimization resulted in novel TTK inhibitors, namely 3-(4-(heterocyclyl)phenyl)-1H-indazole-5-carboxamides. Compounds incorporating the 3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl bicyclic system were potent (TTK IC50 < 10 nM, HCT116 GI50 < 0.1 µM), displayed low off-target activity (>500×), and microsomal stability (T(1/2) > 30 min). A subset was tested in rodent PK and mouse xenograft models of human cancer. Compound 75 (CFI-401870) recapitulated the phenotype of TTK RNAi, demonstrated in vivo tumor growth inhibition upon oral dosing, and was selected for preclinical evaluation.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Neoplasias del Colon/tratamiento farmacológico , Indazoles/química , Indazoles/uso terapéutico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Administración Oral , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Colon/efectos de los fármacos , Colon/enzimología , Colon/patología , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Cristalografía por Rayos X , Femenino , Humanos , Indazoles/administración & dosificación , Indazoles/farmacología , Ratones Desnudos , Modelos Moleculares , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismoRESUMEN
PLK4 was identified as a promising therapeutic target through a systematic approach that combined RNAi screening with gene expression analysis in human breast cancers and cell lines. A drug discovery program culminated in CFI-400945, a potent and selective PLK4 inhibitor. Cancer cells treated with CFI-400945 exhibit effects consistent with PLK4 kinase inhibition, including dysregulated centriole duplication, mitotic defects, and cell death. Oral administration of CFI-400945 to mice bearing human cancer xenografts results in the significant inhibition of tumor growth at doses that are well tolerated. Increased antitumor activity in vivo was observed in PTEN-deficient compared to PTEN wild-type cancer xenografts. Our findings provide a rationale for the clinical evaluation of CFI-400945 in patients with solid tumors, in particular those deficient in PTEN.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/enzimología , Indazoles/farmacología , Indoles/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular , Centriolos/efectos de los fármacos , Centriolos/metabolismo , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The family of Polo-like kinases is important in the regulation of mitotic progression; this work keys on one member, namely Polo-like kinase 4 (PLK4). PLK4 has been identified as a candidate anticancer target which prompted a search for potent and selective inhibitors of PLK4. The body of the paper describes lead generation and optimization work which yielded nanomolar PLK4 inhibitors. Lead generation began with directed virtual screening, using a ligand-based focused library and a PLK4 homology model. Validated hits were used as starting points for the design and discovery of PLK4 inhibitors of novel structure, namely (E)-3-((1H-indazol-6-yl)methylene)indolin-2-ones. Computational models, based on a published X-ray structure (PLK4 kinase domain), were used to understand and optimize the in vitro activity of the series; potent antiproliferative activity was obtained. The kinase selectivity profile and cell cycle analysis of selected inhibitors are described. The results of a xenograft study with an optimized compound 50 (designated CFI-400437) support the potential of these novel PLK4 inhibitors for cancer therapy.
Asunto(s)
Antineoplásicos/síntesis química , Indazoles/síntesis química , Indoles/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Indazoles/química , Indazoles/farmacología , Indoles/química , Indoles/farmacología , Ratones , Ratones SCID , Modelos Moleculares , Estereoisomerismo , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
The Wilms tumor gene (WT1) is mutated or deleted in patients with heredofamilial syndromes associated with the development of Wilms tumors, but is infrequently mutated in sporadic Wilms tumors. By comparing the microarray profiles of syndromic versus sporadic Wilms tumors and WT1-inducible Saos-2 osteosarcoma cells, we identified interferon-inducible protein 16 (IFI16), a transcriptional modulator, as a differentially expressed gene and a candidate WT1 target gene. WT1 induction in Saos-2 osteosarcoma cells led to strong induction of IFI16 expression and its promoter activity was responsive to the WT1 protein. Immunohistochemical analysis showed that IFI16 and WT1 colocalized in WT1-replete Wilms tumors, but not in normal human midgestation fetal kidneys, suggesting that the ability of WT1 to regulate IFI16 in tumors represented an aberrant pathologic relationship. In addition, endogenous IFI16 and WT1 interacted in vivo in two Wilms tumor cell lines. Furthermore, IFI16 augmented the transcriptional activity of WT1 on both synthetic and physiological promoters. Strikingly, short hairpin RNA (shRNA)-mediated knockdown of either IFI16 or WT1 led to decreased growth of Wilms tumor cells. These data suggest that IFI16 and WT1, in certain cellular context including sporadic Wilms tumors, may support cell survival.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Neoplasias Renales/patología , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas WT1/metabolismo , Tumor de Wilms/patología , Neoplasias Óseas/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Perfilación de la Expresión Génica , Humanos , Neoplasias Renales/genética , Osteosarcoma/genética , Transcripción Genética , Regulación hacia Arriba , Proteínas WT1/genética , Tumor de Wilms/genéticaRESUMEN
The STAT proteins are a family of latent transcription factors that are activated by a wide variety of cytokines. Upon receptor engagement, STATs become tyrosine phosphorylated, translocate to the nucleus, and induce expression of target genes. In addition to tyrosine phosphorylation, maximal activation of some STAT proteins requires serine phosphorylation within the transactivation domain. Here we focus on STAT phosphorylation after engagement of the erythropoietin receptor (EPO-R). In Ba/F3-EPO-R cells, EPO induces tyrosine and serine phosphorylation of STAT1, STAT3, STAT5A, and STAT5B. Identical regions of the EPO-R couple to both tyrosine and serine phosphorylation of each cognate STAT protein. A proximal region of the EPO-R lacking cytoplasmic tyrosines couples to STAT1 and STAT3 phosphorylation as well as ERK and p38(HOG) activation, but not JNK/SAPK. STAT1 serine phosphorylation was perturbed by inhibition of ERK and p38 pathways, whereas only inhibition of ERK activation blocked STAT3 serine phosphorylation in response to EPO. STAT5A/B phosphorylation is downstream of EPO-R Tyr(343), however, STAT5A/B serine phosphorylation is unaffected by either ERK or p38 inhibition. Physiological responses induced by EPO may depend on regulation of serine phosphorylation of the STAT molecules by p38(HOG) and the ERK family of kinases as well as additional serine/threonine kinases.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Eritropoyetina/metabolismo , Proteínas de la Leche , Proteínas Proto-Oncogénicas , Serina/metabolismo , Transactivadores/metabolismo , Animales , Western Blotting , Línea Celular , Células Cultivadas , Citoplasma/metabolismo , Humanos , Janus Quinasa 2 , Luciferasas/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Fosforilación , Pruebas de Precipitina , Proteínas Tirosina Quinasas/metabolismo , Receptores de Eritropoyetina/metabolismo , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Factor de Transcripción STAT5 , Células Madre/metabolismo , Factores de Tiempo , Proteínas Supresoras de Tumor , Tirosina/metabolismo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Gain-of-function mutations in exon 3 of beta-catenin (CTNNB1) are specific for Wilms' tumors that have lost WT1, but 50% of WT1-mutant cases lack such "hot spot" mutations. To ask whether stabilization of beta-catenin might be essential after WT1 loss, and to identify downstream target genes, we compared expression profiles in WT1-mutant versus WT1 wild-type Wilms' tumors. Supervised and nonsupervised hierarchical clustering of the expression data separated these two classes of Wilms' tumor. The WT1-mutant tumors overexpressed genes encoding myogenic and other transcription factors (MOX2, LBX1, SIM2), signaling molecules (TGFB2, FST, BMP2A), extracellular Wnt inhibitors (WIF1, SFRP4), and known beta-catenin/TCF targets (FST, CSPG2, CMYC). Beta-Catenin/TCF target genes were overexpressed in the WT1-mutant tumors even in the absence of CTNNB1 exon 3 mutations, and complete sequencing revealed gain-of-function mutations elsewhere in the CTNNB1 gene in some of these tumors, increasing the overall mutation frequency to 75%. Lastly, we identified and validated a novel direct beta-catenin target gene, GAD1, among the WT1-mutant signature genes. These data highlight two molecular classes of Wilms' tumor, and indicate strong selection for stabilization of beta-catenin in the WT1-mutant class. Beta-Catenin stabilization can initiate tumorigenesis in other systems, and this mechanism is likely critical in tumor formation after loss of WT1.