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1.
Chemistry ; 21(18): 6921-9, 2015 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-25784522

RESUMEN

Two small-molecule-drug conjugates (SMDCs, 6 and 7) featuring lysosomally cleavable linkers (namely the Val-Ala and Phe-Lys peptide sequences) were synthesized by conjugation of the αvß3-integrin ligand cyclo[DKP-RGD]-CH2NH2 (2) to the anticancer drug paclitaxel (PTX). A third cyclo[DKP-RGD]-PTX conjugate with a nonpeptide "uncleavable" linker (8) was also synthesized to be tested as a negative control. These three SMDCs were able to inhibit biotinylated vitronectin binding to the purified αVß3-integrin receptor at nanomolar concentrations and showed good stability at pH 7.4 and pH 5.5. Cleavage of the two peptide linkers was observed in the presence of lysosomal enzymes, whereas conjugate 8, which possesses a nonpeptide "uncleavable" linker, remained intact under these conditions. The antiproliferative activities of the conjugates were evaluated against two isogenic cell lines expressing the integrin receptor at different levels: the acute lymphoblastic leukemia cell line CCRF-CEM (αVß3-) and its subclone CCRF-CEM αVß3 (αVß3+). Fairly effective integrin targeting was displayed by the cyclo[DKP-RGD]-Val-Ala-PTX conjugate (6), which was found to differentially inhibit proliferation in antigen-positive CCRF-CEM αVß3 versus antigen-negative isogenic CCRF-CEM cells. The total lack of activity displayed by the "uncleavable" cyclo[DKP-RGD]-PTX conjugate (8) clearly demonstrates the importance of the peptide linker for achieving the selective release of the cytotoxic payload.


Asunto(s)
Antineoplásicos/síntesis química , Dicetopiperazinas/química , Lisosomas/química , Oligopéptidos/química , Paclitaxel/análogos & derivados , Péptidos Cíclicos/síntesis química , Peptidomiméticos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Unión Competitiva , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Liberación de Fármacos , Estabilidad de Medicamentos , Humanos , Integrina alfaVbeta3/metabolismo , Ligandos , Estructura Molecular , Paclitaxel/síntesis química , Paclitaxel/química , Paclitaxel/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Peptidomiméticos/química , Peptidomiméticos/farmacología
2.
Bioorg Med Chem ; 22(15): 4135-50, 2014 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-24980703

RESUMEN

In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.


Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Quinazolinas/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Quinazolinas/síntesis química , Quinazolinas/farmacología , Relación Estructura-Actividad
3.
Bioorg Med Chem ; 21(22): 7047-63, 2013 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-24100158

RESUMEN

Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a fragment based drug discovery approach (FBDD) and subsequent optimization with a combination of structure guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovarian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile.


Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Isoxazoles/química , Isoxazoles/farmacología , Animales , Antineoplásicos/uso terapéutico , Sitios de Unión , Biomarcadores de Tumor/metabolismo , Dominio Catalítico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Isoxazoles/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Unión Proteica/efectos de los fármacos , Relación Estructura-Actividad , Trasplante Heterólogo
4.
Nat Chem Biol ; 4(6): 357-65, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18469809

RESUMEN

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.


Asunto(s)
Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Replicación del ADN/efectos de los fármacos , ADN/efectos de los fármacos , Piperidonas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirroles/farmacología , Animales , Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Células HeLa , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Estructura Molecular , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Fosforilación , Piperidonas/química , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Pirroles/química , Ratas , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Bioorg Med Chem Lett ; 20(22): 6489-94, 2010 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-20932759

RESUMEN

A series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives was optimized as Polo-like kinase 1 inhibitors. Extensive SAR afforded a highly potent and selective PLK1 compound. The compound showed good antiproliferative activity when tested in a panel of tumor cell lines with PLK1 related mechanism of action and with good in vivo antitumor efficacy in two xenograft models after i.v. administration.


Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Quinazolinas/química , Quinazolinas/farmacología , Animales , Línea Celular Tumoral , Humanos , Relación Estructura-Actividad , Trasplante Heterólogo , Quinasa Tipo Polo 1
6.
Comb Chem High Throughput Screen ; 11(7): 523-36, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18694389

RESUMEN

High-content analysis (HCA) is a term used to describe techniques involving multiplexed analysis of fluorescent markers to measure multiple cellular responses to biological stimuli or drug treatment. HCA is usually based on automated microscopy or related technologies, and its value lies in providing multiparametric information on single cells within a population. During the last decade, several HCA approaches have been developed and applied to assess cellular mechanism of action of pharmacologically relevant compounds identified through biochemical screening or similar in vitro methods. With automation and instrument development, these approaches have evolved to the extent that the technique is now routinely used in screening applications, including primary HTS on compound collections. Here, we review the field and discuss in particular the application of HCA to the discovery of small molecule inhibitors targeting kinases which are implicated in Oncology.


Asunto(s)
Bioensayo/métodos , Células/enzimología , Evaluación Preclínica de Medicamentos/métodos , Fosfotransferasas/antagonistas & inhibidores , Fosfotransferasas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Colorantes Fluorescentes
7.
Clin Cancer Res ; 12(13): 4080-9, 2006 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-16818708

RESUMEN

PURPOSE: Aurora kinases play critical roles during mitosis in chromosome segregation and cell division. The aim of this study was to determine the preclinical profile of a novel, highly selective Aurora kinase inhibitor, PHA-680632, as a candidate for anticancer therapy. EXPERIMENTAL DESIGN: The activity of PHA-680632 was assayed in a biochemical ATP competitive kinase assay. A wide panel of cell lines was evaluated for antiproliferative activity. Cell cycle analysis. Immunohistochemistry, Western blotting, and Array Scan were used to follow mechanism of action and biomarker modulation. Specific knockdown of the targets by small interfering RNA was followed to validate the observed phenotypes. Efficacy was determined in different xenograft models and in a transgenic animal model of breast cancer. RESULTS: PHA-680632 is active on a wide range of cancer cell lines and shows significant tumor growth inhibition in different animal tumor models at well-tolerated doses. The mechanism of action of PHA-680632 is in agreement with inhibition of Aurora kinases. Histone H3 phosphorylation in Ser10 is mediated by Aurora B kinase, and our kinetic studies on its inhibition by PHA-680632 in vitro and in vivo show that phosphorylation of histone H3 is a good biomarker to follow activity of PHA-680632. CONCLUSIONS: PHA-680632 is the first representative of a new class of Aurora inhibitors with a high potential for further development as an anticancer therapeutic. On treatment, different cell lines respond differentially, suggesting the absence of critical cell cycle checkpoints that could be the basis for a favorable therapeutic window.


Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirroles/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Aurora Quinasa B , Aurora Quinasas , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Células HL-60 , Células HeLa , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Transgénicos , Estructura Molecular , Fenotipo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/uso terapéutico , Pirroles/uso terapéutico , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos
8.
Cancer Res ; 64(19): 7110-6, 2004 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-15466207

RESUMEN

Cdc7 is an evolutionarily conserved kinase that regulates S phase by promoting replication origin activation. Down-regulation of Cdc7 by small interfering RNA in a variety of tumor cell lines causes an abortive S phase, leading to cell death by either p53-independent apoptosis or aberrant mitosis. Unlike replication fork blockade, Cdc7-depleted tumor cells do not elicit a robust checkpoint response; thus, inhibitory signals preventing additional cell cycle progression are not generated. In normal fibroblasts, however, a p53-dependent pathway actively prevents progression through a lethal S phase in the absence of sufficient Cdc7 kinase. We show that in this experimental system, p53 is required for the lasting maintenance of this checkpoint and for cell viability. With this work we reveal and begin to characterize a novel mechanism that regulates DNA synthesis in human cells, and we suggest that inhibition of Cdc7 kinase represents a promising approach for the development of a new generation of anticancer agents.


Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Fase S/fisiología , Proteína p53 Supresora de Tumor/fisiología , Apoptosis/fisiología , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Progresión de la Enfermedad , Regulación hacia Abajo , Células HeLa , Humanos , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Transfección
9.
Drug Discov Today ; Suppl: 31-42, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-23570164

RESUMEN

High-throughput cell-based assays are becoming a powerful approach in the drug discovery process. The ArrayScan high-content screening (HCS) reader is a cytometer based on a fully automated fluorescence microscope that is able to obtain quantitative information on the intensity and localization of fluorescence signals within single cells over a wide cell population. The aim of this work was to set up an automated HCS multiparameter analysis for the quantification of the in vitro proliferation index of normal human dermal fibroblast (NHDF) cultures. The authors stimulated starved NHDF with insulin-like growth factor-1, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, or serum, and they quantified the proliferation index by measuring the expression of Ki-67 antigen, the incorporation of bromodeoxyuridine (BrdU), and the phosphorylation of the retinoblastoma protein (pRb). This approach also allowed quantification of the mitotic index by phospho-histone H3 staining and the percentage of cells in the S-phase by BrdU incorporation. The proliferation data from the ArrayScan assays were validated by comparison with a reference enzyme-linked immunosorbent assay (ELISA) and by flow cytometry. The measured proliferation indices were highly reproducible in repeated measures and independent experiments. The authors therefore propose that the ArrayScan HCS system could be used for high-throughput multiparameter analysis and quantification of the proliferation of cellular cultures.


Asunto(s)
Proliferación Celular , Fibroblastos/fisiología , Ensayos Analíticos de Alto Rendimiento/instrumentación , División Celular , Células Cultivadas , Citometría de Flujo , Humanos , Índice Mitótico , Fase S , Piel/citología
10.
J Med Chem ; 58(17): 6875-98, 2015 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-26222319

RESUMEN

The nuclear protein poly(ADP-ribose) polymerase-1 (PARP-1) has a well-established role in the signaling and repair of DNA and is a prominent target in oncology, as testified by the number of candidates in clinical testing that unselectively target both PARP-1 and its closest isoform PARP-2. The goal of our program was to find a PARP-1 selective inhibitor that would potentially mitigate toxicities arising from cross-inhibition of PARP-2. Thus, an HTS campaign on the proprietary Nerviano Medical Sciences (NMS) chemical collection, followed by SAR optimization, allowed us to discover 2-[1-(4,4-difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118, 20by). NMS-P118 proved to be a potent, orally available, and highly selective PARP-1 inhibitor endowed with excellent ADME and pharmacokinetic profiles and high efficacy in vivo both as a single agent and in combination with Temozolomide in MDA-MB-436 and Capan-1 xenograft models, respectively. Cocrystal structures of 20by with both PARP-1 and PARP-2 catalytic domain proteins allowed rationalization of the observed selectivity.


Asunto(s)
Antineoplásicos/química , Isoindoles/química , Piperidinas/química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Disponibilidad Biológica , Proliferación Celular/efectos de los fármacos , Dacarbazina/administración & dosificación , Dacarbazina/análogos & derivados , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Xenoinjertos , Ensayos Analíticos de Alto Rendimiento , Humanos , Isoindoles/administración & dosificación , Isoindoles/farmacología , Ratones Endogámicos BALB C , Ratones Desnudos , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Trasplante de Neoplasias , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Piperidinas/administración & dosificación , Piperidinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ratas Sprague-Dawley , Relación Estructura-Actividad , Temozolomida , Neoplasias de la Mama Triple Negativas
11.
FEBS Lett ; 574(1-3): 13-9, 2004 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-15358532

RESUMEN

P84N5 is a death domain containing protein that interacts with the tumor suppressor retinoblastoma protein and induces apoptosis. We cloned and characterized two novel alternatively spliced versions of p84N5. The p84N5 short isoform (p84N5s) lacks the death domain and does not induce apoptosis. We showed that p84N5, but not p84N5s, is cell cycle regulated. We found that p84N5-GFP chimera can rapidly shuttle between the nucleus and the cytoplasm. Taken together, these observations suggest that p84N5 may transmit signals from the nucleus to cytoplasmic effectors.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Proteínas de Unión al ADN , Humanos , Reacción en Cadena de la Polimerasa , Transporte de Proteínas , Proteínas de Unión al ARN
12.
Angiogenesis ; 1(1): 102-116, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-14517397

RESUMEN

Basic fibroblast growth factor (FGF-2) is expressed in vascular endothelium during tumor neovascularization and angioproliferative diseases, including vascular tumors and Kaposi's sarcoma (KS). We have investigated the in vivo biological consequences of endothelial cell activation by endogenous FGF-2 in a mouse aortic endothelial cell line transfected with a retroviral expression vector harboring a human FGF-2 cDNA and the neomycin resistance gene. FGF-2 transfectants, named pZipbFGF2-MAE cells, caused the rapid growth of highly vascularized, non-infiltrating tumors when injected in nude mice. In contrast, lesions grew poorly when cells were injected in immunocompetent syngeneic animals. Histologically, the tumors had the appearance of hemangioendothelioma with spindled areas resembling KS and with numerous CD31+ blood vessels and lacunae. Southern blot analysis of tumor DNA, as well as disaggregation of the lesion followed by in vitro cell culture, revealed that less than 10% of the cells in the tumor mass retain FGF-2 overexpression and neomycin resistance at 6-8 weeks post-injection. Nevertheless, in vitro G418 selection allowed the isolation from the tumor of a FGF-2-overexpressing cell population showing biochemical and biological characteristics similar to those of pZipbFGF2-MAE cells, including the capacity to originate vascular lesions when re-injected in nude mice. To evaluate the effect of angiostatic compounds on the growth and vascularization of pZipbFGF2-MAE cell-induced lesions, nude mice were treated weekly (100mg/kg, i.p.) with the angiostatic sulfonated distamycin A derivative 2,2'-(carbonyl-bis-[imino-N-methyl-4,2-pyrrole carbonyl-imino-{N-methyl-4,2-pyrrole}carbonylimino])-bis-(1,5-naphthalene) disulfonic acid (PNU 153429). The results demonstrate that PNU 153429 inhibits the growth of the lesions and causes a approximately 50% decrease in CD31+ microvessel density. In conclusion, the data indicate that FGF-2-overexpressing endothelial cells cause vascular lesions in immunodeficient mice which may represent a novel model for opportunistic vascular tumors suitable for the evaluation of angiostatic compounds.

13.
J Biomol Screen ; 9(3): 232-43, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15146854

RESUMEN

High-throughput cell-based assays are becoming a powerful approach in the drug discovery process. The ArrayScan high-content screening (HCS) reader is a cytometer based on a fully automated fluorescence microscope that is able to obtain quantitative information on the intensity and localization of fluorescence signals within single cells over a wide cell population. The aim of this work was to set up an automated HCS multiparameter analysis for the quantification of the in vitro proliferation index of normal human dermal fibroblast (NHDF) cultures. The authors stimulated starved NHDF with insulin-like growth factor-1, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, or serum, and they quantified the proliferation index by measuring the expression of Ki-67 antigen, the incorporation of bromodeoxyuridine (BrdU), and the phosphorylation of the retinoblastoma protein (pRb). This approach also allowed quantification of the mitotic index by phospho-histone H3 staining and the percentage of cells in the S-phase by BrdU incorporation. The proliferation data from the ArrayScan assays were validated by comparison with a reference enzyme-linked immunosorbent assay (ELISA) and by flow cytometry. The measured proliferation indices were highly reproducible in repeated measures and independent experiments. The authors therefore propose that the ArrayScan HCS system could be used for high-throughput multiparameter analysis and quantification of the proliferation of cellular cultures.


Asunto(s)
Dermis/citología , Fibroblastos/citología , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Bromodesoxiuridina/farmacocinética , División Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Factor de Crecimiento Epidérmico/farmacología , Factores de Crecimiento de Fibroblastos/farmacología , Fibroblastos/efectos de los fármacos , Humanos , Procesamiento de Imagen Asistido por Computador , Factor I del Crecimiento Similar a la Insulina/farmacología , Antígeno Ki-67/análisis , Mitosis , Índice Mitótico , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Reproducibilidad de los Resultados , Proteína de Retinoblastoma/metabolismo , Fase S
14.
Chempluschem ; 78(7): 663-669, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31986630

RESUMEN

The introduction of a hydrophobic group at position 7 of 9-fluorenone-2-carboxylic acid generates new tubulin binders, the design of which is suggested by modeling studies. The synthesis is based on the use of 2,7-dibromo-fluorenone as starting material. The antiproliferative activity on two different cell lines, fluorescent microscopy, flow cytometry, and sedimentation assay tests confirmed the supposed mechanism.

15.
Clin Cancer Res ; 19(13): 3520-32, 2013 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-23674492

RESUMEN

PURPOSE: Recent developments of second generation Hsp90 inhibitors suggested a potential for development of this class of molecules also in tumors that have become resistant to molecular targeted agents. Disease progression is often due to brain metastases, sometimes related to insufficient drug concentrations within the brain. Our objective was to identify and characterize a novel inhibitor of Hsp90 able to cross the blood-brain barrier (BBB). EXPERIMENTAL DESIGN: Here is described a detailed biochemical and crystallographic characterization of NMS-E973. Mechanism-based anticancer activity was described in cell models, including models of resistance to kinase inhibitors. Pharmacokinetics properties were followed in plasma, tumor, liver, and brain. In vivo activity and pharmacodynamics, as well as the pharmacokinetic/pharmacodynamic relationships, were evaluated in xenografts, including an intracranially implanted melanoma model. RESULTS: NMS-E973, representative of a novel isoxazole-derived class of Hsp90 inhibitors, binds Hsp90α with subnanomolar affinity and high selectivity towards kinases, as well as other ATPases. It possesses potent antiproliferative activity against tumor cell lines and a favorable pharmacokinetic profile, with selective retention in tumor tissue and ability to cross the BBB. NMS-E973 induces tumor shrinkage in different human tumor xenografts, and is highly active in models of resistance to kinase inhibitors. Moreover, consistent with its brain penetration, NMS-E973 is active also in an intracranially implanted melanoma model. CONCLUSIONS: Overall, the efficacy profile of NMS-E973 suggests a potential for development in different clinical settings, including tumors that have become resistant to molecular targeted agents, particularly in cases of tumors which reside beyond the BBB.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/secundario , Resistencia a Antineoplásicos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Isoxazoles/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Sitios de Unión , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/química , Humanos , Concentración 50 Inhibidora , Isoxazoles/química , Isoxazoles/farmacocinética , Ratones , Conformación Molecular , Simulación del Acoplamiento Molecular , Metástasis de la Neoplasia , Especificidad de Órganos/efectos de los fármacos , Unión Proteica , Proteolisis/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
16.
Mol Cancer Ther ; 11(4): 1006-16, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22319201

RESUMEN

Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase considered to be the master player of cell-cycle regulation during mitosis. It is indeed involved in centrosome maturation, bipolar spindle formation, chromosome separation, and cytokinesis. PLK1 is overexpressed in a variety of human tumors and its overexpression often correlates with poor prognosis. Although five different PLKs are described in humans, depletion or inhibition of kinase activity of PLK1 is sufficient to induce cell-cycle arrest and apoptosis in cancer cell lines and in xenograft tumor models. NMS-P937 is a novel, orally available PLK1-specific inhibitor. The compound shows high potency in proliferation assays having low nanomolar activity on a large number of cell lines, both from solid and hematologic tumors. NMS-P937 potently causes a mitotic cell-cycle arrest followed by apoptosis in cancer cell lines and inhibits xenograft tumor growth with clear PLK1-related mechanism of action at well-tolerated doses in mice after oral administration. In addition, NMS-P937 shows potential for combination in clinical settings with approved cytotoxic drugs, causing tumor regression in HT29 human colon adenocarcinoma xenografts upon combination with irinotecan and prolonged survival of animals in a disseminated model of acute myelogenous leukemia in combination with cytarabine. NMS-P937, with its favorable pharmacologic parameters, good oral bioavailability in rodent and nonrodent species, and proven antitumor activity in different preclinical models using a variety of dosing regimens, potentially provides a high degree of flexibility in dosing schedules and warrants investigation in clinical settings.


Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Neoplasias Colorrectales/tratamiento farmacológico , Leucemia/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pirazoles/farmacología , Quinazolinas/farmacología , Administración Oral , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Perros , Femenino , Células HL-60 , Haplorrinos , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patología , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Ratas , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Tipo Polo 1
17.
Anticancer Res ; 30(12): 4973-85, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21187478

RESUMEN

Polo-like kinase 1 (PLK1) is the master regulator of mitosis and a target for anticancer therapy. To develop a marker of PLK1 activity in cells and tumour tissues, this study focused on translational controlled tumour protein (TCTP) and identified serine 46 as a site phosphorylated by PLK1 in vitro. Using an antibody raised against phospho-TCTP-Ser46, it was demonstrated that phosphorylation at this site correlates with PLK1 level and kinase activity in cells. Moreover, PLK1 depletion by siRNA or inactivation by specific inhibitors caused a correspondent decrease in phospho-TCTP-Ser46 signal validating this site as a direct marker of PLK1. Using this marker, the study characterized PLK1 inhibitors in cells by setting up a high-content assay and finally immunohistochemical assay suitable for following inhibitor activity in preclinical tumour models and possibly in clinical studies was developed.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Neoplasias Óseas/enzimología , Neoplasias Óseas/metabolismo , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Femenino , Células HCT116 , Humanos , Ratones , Ratones Desnudos , Osteosarcoma/enzimología , Osteosarcoma/metabolismo , Neoplasias Ováricas/enzimología , Neoplasias Ováricas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Proteína Tumoral Controlada Traslacionalmente 1 , Quinasa Tipo Polo 1
18.
Cancer Res ; 70(24): 10255-64, 2010 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-21159646

RESUMEN

MPS1 kinase is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. It has been found aberrantly overexpressed in a wide range of human tumors and is necessary for tumoral cell proliferation. Here we report the identification and characterization of NMS-P715, a selective and orally bioavailable MPS1 small-molecule inhibitor, which selectively reduces cancer cell proliferation, leaving normal cells almost unaffected. NMS-P715 accelerates mitosis and affects kinetochore components localization causing massive aneuploidy and cell death in a variety of tumoral cell lines and inhibits tumor growth in preclinical cancer models. Inhibiting the SAC could represent a promising new approach to selectively target cancer cells.


Asunto(s)
Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Mitosis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Quinazolinas/farmacología , Huso Acromático/efectos de los fármacos , Aneuploidia , Animales , Antineoplásicos/química , Proteínas de Ciclo Celular/química , Procesos de Crecimiento Celular/efectos de los fármacos , Células HCT116 , Células HeLa , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Terapia Molecular Dirigida/métodos , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas , Ensayos Antitumor por Modelo de Xenoinjerto
19.
J Med Chem ; 52(14): 4380-90, 2009 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-19555113

RESUMEN

Cdc7 kinase has recently emerged as an attractive target for cancer therapy and low-molecular-weight inhibitors of Cdc7 kinase have been found to be effective in the inhibition of tumor growth in animal models. In this paper, we describe synthesis and structure-activity relationships of new 1H-pyrrolo[2,3-b]pyridine derivatives identified as inhibitors of Cdc7 kinase. Progress from (Z)-2-phenyl-5-(1H-pyrrolo[2,3-b]pyridin-3-ylmethylene)-3,5-dihydro-4H-imidazol-4-one (1) to [(Z)-2-(benzylamino)-5-(1H-pyrrolo[2,3-b]pyridin-3-ylmethylene)-1,3-thiazol-4(5H)-one] (42), a potent ATP mimetic inhibitor of Cdc7 kinase with IC(50) value of 7 nM, is also reported.


Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/síntesis química , Piridinas/farmacología , Proteínas de Ciclo Celular/química , Línea Celular , Humanos , Modelos Moleculares , Conformación Molecular , Inhibidores de Proteínas Quinasas/análogos & derivados , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Piridinas/química , Relación Estructura-Actividad
20.
J Biol Chem ; 282(34): 24554-62, 2007 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-17565993

RESUMEN

Increased levels of the repressor element 1/neuron restrictive silencer element (RE1/NRSE) silencing activity promoter, and a consequent reduction in the transcription of many RE1/NRSE-bearing neuronal genes, including brain-derived neurotrophic factor (BDNF), have been demonstrated in Huntington disease (HD) and represent one possible effector of its selective neuronal vulnerability. Restoring the expression levels of neuronal genes in diseased neurons therefore seems to be an attractive therapeutic approach. To this end, we have developed a cell-based reporter assay for monitoring RE1/NRSE silencing activity and validated it by genetically inactivating the RE1/NRSE or pharmacologically stimulating global transcription. In a pilot compound screen, we identified three closely related structural analogues that up-regulate reporter expression at low nanomolar concentrations, and follow-up studies have shown that they efficaciously increase endogenous BDNF levels in HD cells. Moreover, one of the compounds increases the viability of HD cells. Our findings suggest a new avenue for the development of drugs for HD and other neurodegenerative disorders based on the pharmacological up-regulation of the production of the neuronal survival factor BDNF and of other RE1/NRSE-regulated neuronal genes.


Asunto(s)
Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Silenciador del Gen , Proteína Huntingtina , Inmunohistoquímica , Luciferasas/metabolismo , Modelos Químicos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Péptidos/química , Ratas , Factores de Transcripción/metabolismo
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