The advantages offered by established antibiotics in the treatment of infectious diseases are endangered due to the increase in the number of antibiotic-resistant bacterial strains. This leads to a need for new antibacterial compounds. Recently, we discovered a series of compounds termed lipophosphonoxins (LPPOs) that exhibit selective cytotoxicity towards Gram-positive bacteria that include pathogens and resistant strains. For further development of these compounds, it was necessary to identify the mechanism of their action and characterize their interaction with eukaryotic cells/organisms in more detail. Here, we show that at their bactericidal concentrations LPPOs localize to the plasmatic membrane in bacteria but not in eukaryotes. In an in vitro system we demonstrate that LPPOs create pores in the membrane. This provides an explanation of their action in vivo where they cause serious damage of the cellular membrane, efflux of the cytosol, and cell disintegration. Further, we show that (i) LPPOs are not genotoxic as determined by the Ames test, (ii) do not cross a monolayer of Caco-2 cells, suggesting they are unable of transepithelial transport, (iii) are well tolerated by living mice when administered orally but not peritoneally, and (iv) are stable at low pH, indicating they could survive the acidic environment in the stomach. Finally, using one of the most potent LPPOs, we attempted and failed to select resistant strains against this compound while we were able to readily select resistant strains against a known antibiotic, rifampicin. In summary, LPPOs represent a new class of compounds with a potential for development as antibacterial agents for topical applications and perhaps also for treatment of gastrointestinal infections.

Department of Genetics and Microbiology Faculty of Science Charles University Prague Viničná 5 128 43 Prague 2 Czech Republic

Department of Microbiology Faculty of Medicine and Dentistry Palacký University Olomouc Hněvotínská 3 775 15 Olomouc Czech Republic

Division of Biomolecular Physics Institute of Physics Faculty of Mathematics and Physics Charles University Prague Ke Karlovu 5 121 16 Prague 2 Czech Republic

Institute of Microbiology Czech Academy of Sciences v v i Vídeňská 1083 142 20 Prague 4 Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences v v i Flemingovo nám 2 166 10 Prague 6 Czech Republic

TRIOS Ltd Zakouřilova 142 Prague 4 149 00 Prague Czech Republic

University of Chemistry and Technology Technická 5 166 28 Prague Czech Republic

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