Finding effective antibiotics against multi-resistant strains of bacteria has been a challenging race. Linker-Evolved-Group-Optimized-Lipophosphonoxins (LEGO-LPPOs) are small modular synthetic antibacterial compounds targeting the cytoplasmic membrane. Here we focused on understanding the reasons for the variable efficacy of selected LEGO-LPPOs (LEGO-1, LEGO-2, LEGO-3, and LEGO-4) differing in hydrophobic and linker module structure and length. LEGO-1-4 permeabilized cytoplasmic membrane of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli, LEGO-1 with the longest linker module being the most effective. Gram-positive bacteria were more sensitive to LEGO-LPPO action compared to Gram-negatives, which was manifested as a delayed membrane permeabilization, higher minimal inhibitory concentration and lower amount of LEGO-LPPO bound to the cells. Outer membrane permeability measurements and time-kill assay showed that presence of the intact outer membrane brought about reduced susceptibility of Gram-negatives. Using liposome leakage and in silico simulations, we showed that membranes with major content of phosphatidylethanolamine were more prone to LEGO-LPPO permeabilization. The proposed mechanism stems from an electrostatic repulsion between highly positively charged LEGO-1 molecules and positively charged amino groups of phosphatidylethanolamine which destabilizes the membrane. Collectively, these data suggest that LEGO-LPPO membrane activity is enhanced by presence of phosphatidylethanolamine but hindered by presence of intact outer membrane.

• MeSH
• antibakteriální látky * farmakologie   chemie   MeSH
• buněčná membrána metabolismus   MeSH
• Escherichia coli metabolismus   účinky léků   MeSH
• fosfatidylethanolaminy * chemie   metabolismus   MeSH
• mikrobiální testy citlivosti * MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• Staphylococcus aureus účinky léků   metabolismus   MeSH
• vnější bakteriální membrána metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky * MeSH
• fosfatidylethanolaminy * MeSH
• phosphatidylethanolamine MeSH Prohlížeč

Agrimonia eupatoria L. (AE) has a rich tradition of use in wound healing improvement across various cultures worldwide. In previous studies, we revealed that Agrimonia eupatoria L. water extract (AE) possesses a rich polyphenolic composition, displaying remarkable antioxidant properties. Our investigations also demonstrated that lipophosphonoxin (LPPO) exhibited antibacterial efficacy in vitro while preserving the proliferation and differentiation of fibroblasts and keratinocytes. Building upon our prior findings, in this study, we intended to examine whether a combination of AE and LPPO could enhance skin wound healing while retaining antibacterial attributes. The antibacterial activity of AE/LPPO against Staphylococcus aureus was evaluated, alongside its effects on fibroblast-to-myofibroblast transition, the formation of extracellular matrix (ECM), and endothelial cells and keratinocyte proliferation/phenotype. We also investigated AE/LPPO's impact on TGF-β1 and VEGF-A signaling in keratinocytes/fibroblasts and endothelial cells, respectively. Additionally, wound healing progression in rats was examined through macroscopic observation and histological analysis. Our results indicate that AE/LPPO promotes myofibroblast-like phenotypic changes and augments ECM deposition. Clinically relevant, the AE/LPPO did not disrupt TGF-β1 and VEGF-A signaling and accelerated wound closure in rats. Notably, while AE and LPPO individually exhibited antibacterial activity, their combination did not lead to synergism, rather decreasing antibacterial activity, warranting further examination. These findings underscore substantial wound healing improvement facilitated by AE/LPPO, requiring further exploration in animal models closer to human physiology.

• Klíčová slova
• extracellular matrix, phytotherapy, regeneration, repair, skin tissue,
• MeSH
• Agrimonia * chemie   MeSH
• antibakteriální látky * farmakologie   chemie   MeSH
• fibroblasty účinky léků   metabolismus   MeSH
• hojení ran * účinky léků   MeSH
• keratinocyty účinky léků   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• potkani Sprague-Dawley MeSH
• proliferace buněk účinky léků   MeSH
• rostlinné extrakty * farmakologie   chemie   MeSH
• Staphylococcus aureus * účinky léků   MeSH
• transformující růstový faktor beta1 metabolismus   MeSH
• vaskulární endoteliální růstový faktor A metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky * MeSH
• rostlinné extrakty * MeSH
• transformující růstový faktor beta1 MeSH
• vaskulární endoteliální růstový faktor A MeSH

In the past few decades, society has faced rapid development and spreading of antimicrobial resistance due to antibiotic misuse and overuse and the immense adaptability of bacteria. Difficulties in obtaining effective antimicrobial molecules from natural sources challenged scientists to develop synthetic molecules with antimicrobial effect. We developed modular molecules named LEGO-Lipophosphonoxins (LEGO-LPPO) capable of inducing cytoplasmic membrane perforation. In this structure-activity relationship study we focused on the role of the LEGO-LPPO hydrophobic module directing the molecule insertion into the cytoplasmic membrane. We selected three LEGO-LPPO molecules named C9, C8 and C7 differing in the length of their hydrophobic chain and consisting of an alkenyl group containing one double bond. The molecule with the long hydrophobic chain (C9) was shown to be the most effective with the lowest MIC and highest perforation rate both in vivo and in vitro. We observed high antimicrobial activity against both G+ and G- bacteria with significant differences in LEGO-LPPOs mechanism of action on these two cell types. We observed a highly cooperative mechanism of LEGO-LPPO action on G- bacteria as well as on liposomes resembling G- bacteria. LEGO-LPPO action on G- bacteria was significantly slower compared to G+ bacteria suggesting the role of the outer membrane in affecting the LEGO-LPPOs perforation rate. This notion was supported by the higher sensitivity of the E. coli strain with a compromised outer membrane. Finally, we noted that the composition of the cytoplasmic membrane affects the activity of LEGO-LPPOs since the presence of phosphatidylethanolamine increases their membrane disrupting activity.

• Publikační typ
• časopisecké články MeSH

Lipophosphonoxins (LPPOs) represent a new group of membrane-targeting antibiotics. Three generations of LPPOs have been described: First-generation LPPOs, second-generation LPPOs, and LEGO-LPPOs. All three generations have a similar mode of bactericidal action of targeting and disrupting the bacterial cytoplasmic membrane of prokaryotic cells, with limited effect on eukaryotic cells. First-generation LPPOs showed excellent bactericidal activity against Gram-positive species, including multiresistant strains. Second-generation LPPOs broaden the antibiotic effect also against Gram-negative bacteria. However, both first- and second-generation LPPOs lose their antibacterial activity in the presence of serum albumin. LEGO-LPPOs were found to be active against both Gram-positive and Gram-negative bacteria, have better selectivity as compared to first- and second-generation resistance to LEGO-LPPOs was also not observed, and are active even in the presence of serum albumin. Second-generation LPPOs have been studied as antimicrobial additives in bone cement and as nanofiber dressing components in the treatment of wound infections in mice. Second-generation LPPOs and LEGO-LPPOs were also tested to treat ex vivo simulated endodontic infections in dental root canals. The results of all these studies were encouraging and suggested further investigation of LPPOs in these indications. This paper aims to review and compile published data on LPPOs.

• Klíčová slova
• LPPOs, Lipophosphonoxins, antibiotics, antimicrobial resistance,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The alarming rise of bacterial antibiotic resistance requires the development of new compounds. Such compounds, lipophosphonoxins (LPPOs), were previously reported to be active against numerous bacterial species, but serum albumins abolished their activity. Here we describe the synthesis and evaluation of novel antibacterial compounds termed LEGO-LPPOs, loosely based on LPPOs, consisting of a central linker module with two attached connector modules on either side. The connector modules are then decorated with polar and hydrophobic modules. We performed an extensive structure-activity relationship study by varying the length of the linker and hydrophobic modules. The best compounds were active against both Gram-negative and Gram-positive species including multiresistant strains and persisters. LEGO-LPPOs act by first depleting the membrane potential and then creating pores in the cytoplasmic membrane. Importantly, their efficacy is not affected by the presence of serum albumins. Low cytotoxicity and low propensity for resistance development demonstrate their potential for therapeutic use.

• MeSH
• albuminy MeSH
• antibakteriální látky * chemie   MeSH
• buněčná membrána MeSH
• gramnegativní bakterie MeSH
• grampozitivní bakterie * MeSH
• mikrobiální testy citlivosti MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• albuminy MeSH
• antibakteriální látky * MeSH

(1) Background: The root canal system has complex anatomical and histological features that make it impossible to completely remove all bacteria by mechanical means only; they must be supplemented with disinfectant irrigation. Current disinfectants are unable to eliminate certain microorganisms that persist in the root canal, resulting in treatment failure. At the Institute of Organic Chemistry and Biochemistry, Prague, novel substances with the bactericidal effect, termed lipophosphonoxins (LPPOs), have been discovered. The aim of this pilot study was to investigate the ex vivo effects of second- and third-generation LPPOs on Enterococcus faecalis and compare them with 5% sodium hypochlorite (NaOCl), 0.12% chlorhexidine digluconate, and 17% ethylenediaminetetraacetic acid (EDTA). (2) Methods: The root canal's dentin was used as a carrier for biofilm formation in the extracted human mature mandibular premolars. The samples were filled with cultivation broth and 0.25% glucose with tested solutions. In control samples, only fresh cultivation broth (negative control) and cultivation broth with bacterial suspension (growth control) were used. Each sample was inoculated with E. faecalis CCM4224 except for the negative control, and cultivation was performed. To determine the number of planktonic cells, the sample content was inoculated on blood agar. To evaluate biofilm formation inhibition, samples were placed in tubes with BHI. (3) Results: LPPOs exhibited a reduction in biofilm growth and bacteria comparable to NaOCl, and they were superior to other tested disinfectants. (4) Conclusions: The study results suggest the effect of lipophosphonoxins on E. faecalis CCM 4224 reduces planktonic bacterial cells and inhibits formation of biofilm in root canal samples.

• Klíčová slova
• E. faecalis, EDTA, biofilm, chlorhexidine digluconate, lipophosphonoxins, root canal, sodium hypochlorite,
• Publikační typ
• časopisecké články MeSH

Active wound dressings are attracting extensive attention in soft tissue repair and regeneration, including bacteria-infected skin wound healing. As the wide use of antibiotics leads to drug resistance we present here a new concept of wound dressings based on the polycaprolactone nanofiber scaffold (NANO) releasing second generation lipophosphonoxin (LPPO) as antibacterial agent. Firstly, we demonstrated in vitro that LPPO released from NANO exerted antibacterial activity while not impairing proliferation/differentiation of fibroblasts and keratinocytes. Secondly, using a mouse model we showed that NANO loaded with LPPO significantly reduced the Staphylococcus aureus counts in infected wounds as evaluated 7 days post-surgery. Furthermore, the rate of degradation and subsequent LPPO release in infected wounds was also facilitated by lytic enzymes secreted by inoculated bacteria. Finally, LPPO displayed negligible to no systemic absorption. In conclusion, the composite antibacterial NANO-LPPO-based dressing reduces the bacterial load and promotes skin repair, with the potential to treat wounds in clinical settings.

• MeSH
• antibakteriální látky aplikace a dávkování   terapeutické užití   MeSH
• hojení ran účinky léků   MeSH
• infekce v ráně farmakoterapie   MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• nanovlákna * MeSH
• obvazy * MeSH
• stafylokokové infekce farmakoterapie   MeSH
• Staphylococcus aureus * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH