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Insights into the Mechanism of Action of Bactericidal Lipophosphonoxins.

Panova N, Zborníková E, Šimák O, Pohl R, Kolář M, Bogdanová K, Večeřová R, Seydlová G, Fišer R, Hadravová R, Šanderová H, Vítovská D, Šiková M, Látal T, Lovecká P, Barvík I, Krásný L, Rejman D - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

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Interaction of fluorescently labeled LPPO DR5823 with model membrane.(A) Excitation (solid line) and emission (dotted line) spectra of DR5823 in buffer (in green) and liposomes (in red). (B) Incorporation of DR5823 to the liposome membrane.
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pone.0145918.g008: Interaction of fluorescently labeled LPPO DR5823 with model membrane.(A) Excitation (solid line) and emission (dotted line) spectra of DR5823 in buffer (in green) and liposomes (in red). (B) Incorporation of DR5823 to the liposome membrane.

Mentions: To provide insights into the mechanism of LPPO interaction with the membrane we designed and synthesized LPPO DR5823 –a fluorescently labeled analog of DR5026 with minimal structural changes to avoid alteration of its physicochemical and biological properties (the structure, synthesis, and antibacterial activity of DR5823 are described in S1 Supporting Information and S1 Table respectively). As model membrane we used dioleoylphosphatidylcholine (DOPC) bilayers. Upon DR5823 addition fluorescence intensity substantially increased (4.5 times in comparison to buffer) (Fig 8A), suggesting LPPO incorporation into the non-polar environment of the membrane. Excitation spectrum showed maxima at 261 and 315 nm and fluorescence emission dominated at 411 nm.


Insights into the Mechanism of Action of Bactericidal Lipophosphonoxins.

Panova N, Zborníková E, Šimák O, Pohl R, Kolář M, Bogdanová K, Večeřová R, Seydlová G, Fišer R, Hadravová R, Šanderová H, Vítovská D, Šiková M, Látal T, Lovecká P, Barvík I, Krásný L, Rejman D - PLoS ONE (2015)

Interaction of fluorescently labeled LPPO DR5823 with model membrane.(A) Excitation (solid line) and emission (dotted line) spectra of DR5823 in buffer (in green) and liposomes (in red). (B) Incorporation of DR5823 to the liposome membrane.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4696656&req=5

pone.0145918.g008: Interaction of fluorescently labeled LPPO DR5823 with model membrane.(A) Excitation (solid line) and emission (dotted line) spectra of DR5823 in buffer (in green) and liposomes (in red). (B) Incorporation of DR5823 to the liposome membrane.
Mentions: To provide insights into the mechanism of LPPO interaction with the membrane we designed and synthesized LPPO DR5823 –a fluorescently labeled analog of DR5026 with minimal structural changes to avoid alteration of its physicochemical and biological properties (the structure, synthesis, and antibacterial activity of DR5823 are described in S1 Supporting Information and S1 Table respectively). As model membrane we used dioleoylphosphatidylcholine (DOPC) bilayers. Upon DR5823 addition fluorescence intensity substantially increased (4.5 times in comparison to buffer) (Fig 8A), suggesting LPPO incorporation into the non-polar environment of the membrane. Excitation spectrum showed maxima at 261 and 315 nm and fluorescence emission dominated at 411 nm.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus