RESUMO
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.
Assuntos
Camptotecina/análogos & derivados , Substâncias Intercalantes/farmacologia , Fenazinas/farmacologia , Animais , Antígenos de Neoplasias , Camptotecina/farmacologia , Catálise/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Análise por Conglomerados , DNA/química , DNA/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Fase G1/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Perfilação da Expressão Gênica , Células HCT116 , Humanos , Substâncias Intercalantes/química , Irinotecano , Masculino , Camundongos , Camundongos Nus , Mitose/efeitos dos fármacos , Mutação , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Fenazinas/química , Transplante Heterólogo , Ensaios Antitumorais Modelo de Xenoenxerto , Leveduras/efeitos dos fármacos , Leveduras/enzimologia , Leveduras/genéticaRESUMO
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.
Assuntos
Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , RNA Polimerase III/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Farmacorresistência Fúngica/genética , Humanos , Dados de Sequência Molecular , Peso Molecular , Mutação/genética , Subunidades Proteicas/genética , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , RNA de Transferência/biossíntese , RNA de Transferência/genética , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/genética , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
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.
