RESUMEN
The activity and stability of the p53 tumor suppressor are regulated by the human homologue of the mouse double minute 2 (Hdm2) oncoprotein. It has been hypothesized that small molecules disrupting the Hdm2:p53 complex would allow for the activation of p53 and result in growth suppression. We have identified small-molecule inhibitors of the Hdm2:p53 interaction using our proprietary ThermoFluor microcalorimetry technology. Medicinal chemistry and structure-based drug design led to the development of an optimized series of benzodiazepinediones, including TDP521252 and TDP665759. Activities were dependent on the expression of wild-type (wt) p53 and Hdm2 as determined by lack of potency in mutant or null p53-expressing cell lines or cells engineered to no longer express Hdm2 and wt p53. TDP521252 and TDP665759 inhibited the proliferation of wt p53-expressing cell lines with average IC(50)s of 14 and 0.7 micromol/L, respectively. These results correlated with the direct cellular dissociation of Hdm2 from wt p53 observed within 15 minutes in JAR choriocarcinoma cells. Additional activities of these inhibitors in vitro include stabilization of p53 protein levels, up-regulation of p53 target genes in a DNA damage-independent manner, and induction of apoptosis in HepG2 cells. Administration of TDP665759 to mice led to an increase in p21(waf1/cip1) levels in liver samples. Finally, TDP665759 synergizes with doxorubicin both in culture and in an A375 xenograft model to decrease tumor growth. Taken together, these data support the potential utility of small-molecule inhibitors of the Hdm2:p53 interaction for the treatment of wt p53-expressing tumors.
Asunto(s)
Benzodiazepinonas/farmacología , Doxorrubicina/farmacología , Proteínas Proto-Oncogénicas c-mdm2/efectos de los fármacos , Proteína p53 Supresora de Tumor/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptosis/efectos de los fármacos , Benzodiazepinas/química , Benzodiazepinas/farmacología , Benzodiazepinonas/administración & dosificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Humanos , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/metabolismo , Ratones , Ratones Desnudos , Complejos Multiproteicos , Mutación , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.
Asunto(s)
Benzodiazepinas/síntesis química , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/agonistas , Benzodiazepinas/química , Benzodiazepinas/farmacología , Sitios de Unión , Línea Celular Tumoral , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Imitación Molecular , Estructura Molecular , Proteínas Proto-Oncogénicas c-mdm2 , Estereoisomerismo , Relación Estructura-Actividad , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
There is increasing evidence that tumor-associated macrophages promote the malignancy of some cancers. Colony-stimulating factor-1 (CSF-1) is expressed by many tumors and is a growth factor for macrophages and mediates osteoclast differentiation. Herein, we report the efficacy of a novel orally active CSF-1 receptor (CSF-1R) kinase inhibitor, JNJ-28312141, in proof of concept studies of solid tumor growth and tumor-induced bone erosion. H460 lung adenocarcinoma cells did not express CSF-1R and were not growth inhibited by JNJ-28312141 in vitro. Nevertheless, daily p.o. administration of JNJ-28312141 caused dose-dependent suppression of H460 tumor growth in nude mice that correlated with marked reductions in F4/80(+) tumor-associated macrophages and with increased plasma CSF-1, a possible biomarker of CSF-1R inhibition. Furthermore, the tumor microvasculature was reduced in JNJ-28312141-treated mice, consistent with a role for macrophages in tumor angiogenesis. In separate studies, JNJ-28312141 was compared with zoledronate in a model in which MRMT-1 mammary carcinoma cells inoculated into the tibias of rats led to severe cortical and trabecular bone lesions. Both agents reduced tumor growth and preserved bone. However, JNJ-28312141 reduced the number of tumor-associated osteoclasts superior to zoledronate. JNJ-28312141 exhibited additional activity against FMS-related receptor tyrosine kinase-3 (FLT3). To more fully define the therapeutic potential of this new agent, JNJ-28312141 was evaluated in a FLT3-dependent acute myeloid leukemia tumor xenograft model and caused tumor regression. In summary, this novel CSF-1R/FLT3 inhibitor represents a new agent with potential therapeutic activity in acute myeloid leukemia and in settings where CSF-1-dependent macrophages and osteoclasts contribute to tumor growth and skeletal events.
Asunto(s)
Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/secundario , Imidazoles/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Animales , Neoplasias Óseas/enzimología , Neoplasias Óseas/patología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Inmunohistoquímica , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Desnudos , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Ratas , Ratas Sprague-Dawley , Receptor de Factor Estimulante de Colonias de Macrófagos/sangre , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Especificidad por Sustrato , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Guided by structure-based drug design, modification of the 1,4-benzodiazepin-2,5-dione lead compound 1 resulted in the discovery of 19, a potent and orally bioavailable antagonist of the HDM2-p53 protein-protein interaction (FP IC50 = 0.7 microM, F approximately 100%).