Target identification and mechanistic studies of cytotoxic agents are challenging processes that are both time-consuming and costly. Here we describe an approach to mechanism of action studies for potential anticancer compounds by utilizing the simple prokaryotic system, E. coli, and we demonstrate its utility with the characterization of a ruthenium polypyridyl complex [Ru(bpy) 2 dmbpy 2+ ]. Expression of the photoconvertible fluorescent protein Dendra2 facilitated both high throughput studies and single-cell imaging. This allowed for simultaneous ratiometric analysis of inhibition of protein production and phenotypic investigations. The profile of protein production, filament size and population, and nucleoid morphology revealed important differences between inorganic agents that damage DNA vs more selective inhibitors of transcription and translation. Trace metal analysis demonstrated that DNA is the preferred nucleic acid target of the ruthenium complex, but further studies in human cancer cells revealed altered cell signaling pathways compared to the commonly administrated anticancer agent cisplatin. This study demonstrates E. coli can be used to rapidly distinguish between compounds with disparate mechanisms of action and also for more subtle distinctions within in studies in mammalian cells.
|Number of pages||13|
|State||Published - Aug 6 2018|
Bibliographical noteFunding Information:
This work is supported by the National Institutes of Health (R01GM107586). YS was the recipient of a University of Kentucky Opportunity Fellowship. All metal uptake studies are done with technical support of Environmental Research Training Laboratories (ERTL) at the University of Kentucky. Experiments were performed at the UK Flow Cytometry & Cell Sorting core facility, which is supported in part by the Office of the Vice President for Research, the Markey Cancer Center, and an NCI Center Core Support Grant (P30 CA177558) to the University of Kentucky Markey Cancer Center.
© 2018 American Chemical Society.
- bacterial cytological profiling
- drug discovery
ASJC Scopus subject areas
- Molecular Medicine
- Pharmaceutical Science
- Drug Discovery