EPR as a probe of the intracellular speciation of ruthenium(III) anticancer compounds

EPR (electron paramagnetic resonance) has been used to study interactions of the Ru(III) anticancer compounds imidazolium [trans-RuCl4(1H-imidazole)(DMSO-S)] (NAMI-A) and indazolium [trans-RuCl4(1H-indazole)(2)] (KP1019) with isolated subcellular components and whole cells of the yeast Saccharomyces cerevisiae. These studies are the first to probe the intracellular speciation of ruthenium using the EPR technique. Initially, NAMI-A and KP1019 were incubated at 30 degrees C, for time periods up to 24 hours with isolated cell wall, mitochondrial, cytoplasmic, and nuclear fractions of S. cerevisiae. EPR measurements demonstrate that NAMI-A initially forms non-coordinate interactions with each cell component. After longer incubation times these are replaced by coordinated species, particularly with cytoplasmic proteins. KP1019 shows a greater tendency to coordinate directly with cell components, demonstrating significant interactions with mitochondria and cytoplasmic proteins. Subsequently, each complex was incubated with whole cells of S. cerevisiae at 30 degrees C and whole-cell EPR measurements detected Ru(III) species in measurable concentrations even after 24 hours of incubation. Analysis of the resulting EPR spectra suggests NAMI-A interacts predominantly with cell walls, while KP1019 was found to be coordinating with both the mitochondrial and cytoplasmic protein fractions. Comparison of the signal intensity of these data with those from incubation with whole cells at 4 degrees C indicates different modes of transmembrane transport for each complex. These studies demonstrate that EPR can provide valuable insight into the oxidation state and speciation of ruthenium compounds in cellular environments.