RESUMEN
MLN944 (XR5944) is a novel bis-phenazine that has demonstrated exceptional efficacy against a number of murine and human tumor models. The drug was reported originally as a dual topoisomerase I/II poison, but a precise mechanism of action for this compound remains to be determined. Several lines of evidence, including the marginal ability of MLN944 to stabilize topoisomerase-dependent cleavage, and the sustained potency of MLN944 in mammalian cells with reduced levels of both topoisomerases, suggest that other activities of the drug exist. In this study, we show that MLN944 intercalates into DNA, but has no effect on the catalytic activity of either topoisomerase I or II. MLN944 displays no significant ability to stimulate DNA scission mediated by either topoisomerase I or II compared with camptothecin or etoposide, respectively. In addition, yeast genetic models also point toward a topoisomerase-independent mechanism of action. To examine cell cycle effects, synchronized human HCT116 cells were treated with MLN944, doxorubicin, camptothecin, or a combination of the latter two to mimic a dual topoisomerase poison. MLN944 treatment was found to induce a G(1) and G(2) arrest in cells that is unlike the typical G(2)-M arrest noted with known topoisomerase poisons. Finally, transcriptional profiling analysis of xenograft tumors treated with MLN944 revealed clusters of regulated genes distinct from those observed in irinotecan hydrochloride (CPT-11)-treated tumors. Taken together, these findings suggest that the primary mechanism of action of MLN944 likely involves DNA binding and intercalation, but does not appear to involve topoisomerase inhibition.
Asunto(s)
Camptotecina/análogos & derivados , Sustancias Intercalantes/farmacología , Fenazinas/farmacología , Animales , Antígenos de Neoplasias , Camptotecina/farmacología , Catálisis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Análisis por Conglomerados , ADN/química , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Fase G1/efectos de los fármacos , Fase G2/efectos de los fármacos , Perfilación de la Expresión Génica , Células HCT116 , Humanos , Sustancias Intercalantes/química , Irinotecán , Masculino , Ratones , Ratones Desnudos , Mitosis/efectos de los fármacos , Mutación , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Fenazinas/química , Trasplante Heterólogo , Ensayos Antitumor por Modelo de Xenoinjerto , Levaduras/efectos de los fármacos , Levaduras/enzimología , Levaduras/genéticaRESUMEN
A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.
Asunto(s)
Antifúngicos/farmacología , Inhibidores Enzimáticos/farmacología , ARN Polimerasa III/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Candida albicans/efectos de los fármacos , Candida albicans/enzimología , Candida albicans/genética , Células Cultivadas , Relación Dosis-Respuesta a Droga , Farmacorresistencia Fúngica/genética , Humanos , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Subunidades de Proteína/genética , ARN Polimerasa III/genética , ARN Polimerasa III/metabolismo , ARN de Transferencia/biosíntesis , ARN de Transferencia/genética , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/genética , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
Although the biochemical targets of most drugs are known, the biological consequences of their actions are typically less well understood. In this study, we have used two whole-genome technologies in Saccharomyces cerevisiae to determine the cellular impact of the proteasome inhibitor PS-341. By combining population genomics, the screening of a comprehensive panel of bar-coded mutant strains, and transcript profiling, we have identified the genes and pathways most affected by proteasome inhibition. Many of these function in regulated protein degradation or a subset of mitotic activities. In addition, we identified Rpn4p as the transcription factor most responsible for the cell's ability to compensate for proteasome inhibition. Used together, these complementary technologies provide a general and powerful means to elucidate the cellular ramifications of drug treatment.