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1.
Curr Res Struct Biol ; 4: 96-105, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35469152

RESUMEN

Clostridium difficile toxins are the primary causative agents for hospital-acquired diarrhea and pseudomembranous colitis. Numerous monoclonal antibodies (mAbs) targeting different domains of Clostridium difficile toxin have been reported. Here we report the crystal structures of two mAbs, B1 and B2, in complex with the glycosyltransferase domain (GTD) of the Clostridium difficile toxin B (TcdB). B2 bound to the N-terminal 4 helix bundle of the GTD, a conserved membrane localization domain (MLD) found in the large clostridial glycosylating toxin family implicated in targeting plasma membrane. B1 bound to a distinct epitope at the hinge region between the MLD and the catalytic subdomain of the GTD. Functional studies revealed the potency of these mAbs in vitro and in vivo to be synergistic when given in combination.

2.
PLoS One ; 7(3): e33823, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22457791

RESUMEN

Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.


Asunto(s)
Apoptosis/fisiología , Ácidos Grasos Monoinsaturados/metabolismo , Neoplasias/patología , Estearoil-CoA Desaturasa/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Estearoil-CoA Desaturasa/fisiología
3.
J Med Chem ; 55(6): 2641-8, 2012 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-22339127

RESUMEN

We have studied the subtleties of fragment docking and binding using data generated in a Pim-1 kinase inhibitor program. Crystallographic and docking data analyses have been undertaken using inhibitor complexes derived from an in-house surface plasmon resonance (SPR) fragment screen, a virtual needle screen, and a de novo designed fragment inhibitor hybrid. These investigations highlight that fragments that do not fill their binding pocket can exhibit promiscuous hydrophobic interactions due to the lack of steric constraints imposed on them by the boundaries of said pocket. As a result, docking modes that disagree with an observed crystal structure but maintain key crystallographically observed hydrogen bonds still have potential value in ligand design and optimization. This observation runs counter to the lore in fragment-based drug design that all fragment elaboration must be based on the parent crystal structure alone.


Asunto(s)
Inhibidores Enzimáticos/química , Modelos Moleculares , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-pim-1/química , Cristalografía por Rayos X , Diseño de Fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Molecular , Unión Proteica , Estereoisomerismo , Relación Estructura-Actividad
4.
Bioorg Med Chem Lett ; 21(10): 3050-6, 2011 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-21507633

RESUMEN

Novel benzofuran-2-carboxylic acids, exemplified by 29, 38 and 39, have been discovered as potent Pim-1 inhibitors using fragment based screening followed by X-ray structure guided medicinal chemistry optimization. The compounds demonstrate potent inhibition against Pim-1 and Pim-2 in enzyme assays. Compound 29 has been tested in the Ambit 442 kinase panel and demonstrates good selectivity for the Pim kinase family. X-ray structures of the inhibitor/Pim-1 binding complex reveal important salt-bridge and hydrogen bond interactions mediated by the compound's carboxylic acid and amino groups.


Asunto(s)
Benzofuranos/química , Ácidos Carboxílicos/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Animales , Ácidos Carboxílicos/síntesis química , Ácidos Carboxílicos/química , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Modelos Moleculares , Estructura Molecular , Ratas
5.
Exp Cell Res ; 316(2): 258-71, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-19732767

RESUMEN

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21(/Cip) and p27(/Kip1). Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.


Asunto(s)
Ciclo Celular/genética , Senescencia Celular/genética , Complejo de la Endopetidasa Proteasomal/deficiencia , Transactivadores/deficiencia , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , ADN/análisis , Células Epiteliales/citología , Células Epiteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Fase G1/genética , Expresión Génica/genética , Células HeLa , Humanos , Fosforilación/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Interferente Pequeño/genética , Fase de Descanso del Ciclo Celular/genética , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Sulfotransferasas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Transfección , Proteínas Ubiquitinadas/metabolismo , Regulación hacia Arriba/genética , beta-Galactosidasa/metabolismo , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismo
6.
Int J Oncol ; 32(4): 739-48, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18360701

RESUMEN

Two of the three components of anthrax toxin, protective antigen (PA) and lethal factor (LF), together known as lethal toxin (LeTx), reportedly show anti-tumor activity in melanoma in vitro and in vivo. The growth inhibitory activity of LeTx in culture was determined in nine human cancer cell lines, including melanoma, neuroblastoma and adenocarcinoma cells, as well as in human umbilical vein endothelial cells (HUVEC). The contribution of the two known PA receptor proteins, ANTXR1/TEM8 and ANTXR2/CMG2, to the sensitivity of the cells was assessed. The efficacy of LeTx was evaluated in vivo in the SK-N-AS neuroblastoma and SK-MEL-28 melanoma tumor xenograft models. Sensitivity to LeTx in vitro was observed in the neuroblastoma and colorectal adenocarcinoma cells and HUVEC, as well as melanoma cells. ANTXR1/TEM8 and ANTXR2/CMG2 protein expression studies suggested that a certain threshold of the PA receptor protein level must be met for sensitivity to LeTx to be observed. However, although the SK-N-AS neuroblastoma cells expressed the highest levels of receptor proteins and achieved the lowest IC50 in vitro (0.1 ng/ml), we observed no correlation between either the ANTXR1/TEM8 or ANTXR2/CMG2 protein levels and sensitivity to LeTx in vitro. In vivo, LeTx was an active anti-tumor agent when administered intravenously to mice bearing the human SK-N-AS or SK-MEL-28 tumor xenografts. The tumor growth delays were 6-8 days with a lower dose regimen and 14-16 days with a higher dose regimen for the two tumor models. These in vitro data suggest that LeTx may have broad therapeutic indications in cancer and the in vivo studies demonstrate that LeTx has systemic efficacy in neuroblastoma as well as melanoma. The therapeutic potential of LeTx needs to be further investigated in non-melanoma tumor models expressing the ANTXR1/TEM8 and/or ANTXR2/CMG2 protein.


Asunto(s)
Antígenos Bacterianos/uso terapéutico , Toxinas Bacterianas/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , Neuroblastoma/tratamiento farmacológico , Receptores de Péptidos/análisis , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Niño , Femenino , Humanos , Masculino , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neuroblastoma/patología , Ensayos Antitumor por Modelo de Xenoinjerto
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