Ru binding to RNA following treatment with the antimetastatic prodrug NAMI-A in Saccharomyces cerevisiae and in vitro

Abstract

[ImH][trans-Ru(III)Cl(4)(DMSO)(Im)] (where DMSO is dimethyl sulfoxide and Im is imidazole) (NAMI-A) is an antimetastatic prodrug currently in phase II clinical trials. The mechanisms of action of this and related Ru-based anticancer agents are not well understood, but several cellular targets have been suggested. Although Ru has been observed to bind to DNA following in vitro NAMI-A exposure, little is known about Ru-DNA interactions in vivo and even less is known about how this or related metallodrugs might influence cellular RNA. In this study, Ru accumulation in cellular RNA was measured following treatment of Saccharomyces cerevisiae with NAMI-A. Drug-dependent growth and cell viability indicate relatively high tolerance, with approximately 40% cell death occurring at 6h for 450μM NAMI-A. Significant dose-dependent accumulation of Ru in cellular RNA was observed by inductively coupled plasma mass spectrometry measurements on RNA extracted from yeast treated with NAMI-A. In vitro, binding of Ru species to drug-treated model DNA and RNA oligonucleotides at pH 6.0 and 7.4 was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the presence and absence of the reductant ascorbate. The extent of Ru-nucleotide interactions increases slightly with lower pH and significantly in the presence of ascorbate, with differences in observed species distribution. Taken together, these studies demonstrate the accumulation of aquated and reduced derivatives of NAMI-A on RNA in vitro and in cellulo, and enhanced binding with nucleic acid targets in a tumorlike acidic, reducing environment. To our knowledge, this is also the first study to characterize NAMI-A treatment of S. cerevisiae, a genetically tractable model organism.