Uncovering the mechanism of action of a novel ruthenium-based antibiotic

Abstract

Historically, metal containing compounds (MCC) have demonstrated significant therapeutic potential both as antimicrobial agents and for cancer treatment. MCCs represent some of the oldest antimicrobial compounds known such as Salvarsan, the first antibiotic discovered. This study investigates a novel ruthenium-based complex (Ru) for its antibacterial properties. Thorough knowledge of the mode of action (MoA) is a crucial step in the development of new antimicrobial compounds. In this study, a multi-assay investigative approach was employed to investigate the MoA of Ru in the Gram-positive model organism Bacillus subtilis. Initial inhibition activity experiments revealed that the Ru compound exhibits a minimum inhibitory concentration (MIC) of 6.25 µM. In this study, bacterial cytological profiling showed indication of broad cellular disturbances. Most notably, a proteome-wide protein degradation effect was observed. Further, the membrane was disturbed, and DNA damage was detected. A novel assay measuring the induced protein degradation with different reactive oxygen species scavengers provided an unexpected and striking result. This provided evidence that oxidative stress, specifically caused by hydroxyl radicals rather than superoxide radicals, are a central part of the MoA. Additional microscopy with a superoxide radical probe further suggests that Ru appears to reduce the intracellular superoxide concentrations. Collectively, these findings indicate a MoA where Ru causes oxidative stress which then leads to macromolecular damage and rapid protein degradation. The new scavenger assay results were particularly interesting and brought compelling insights, in which the presence of a hydroxyl radical scavenger stopped the rapid protein degradation induced by Ru. Understanding this novel MCC, which exhibits this unique rapid protein degradation MoA, could significantly advance the understanding towards innovation of new antibiotics in the important fight against antibiotic resistance.