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Lipophosphonoxins II: Design, Synthesis and Properties of Novel Broad Spectrum Antibacterial Agents
Abstract
The increase in the number of bacterial strains resistant to known antibiotics is alarming. In this study we report the synthesis of novel compounds termed Lipophosphonoxins II (LPPO II). We show that LPPO II display excellent activities against Gram positive and -negative bacteria, including pathogens and multiresistant strains. We describe their mechanism of action - plasmatic membrane pore-forming activity selective for bacteria. Importantly, LPPO II neither damage nor cross the eukaryotic plasmatic membrane at their bactericidal concentrations. Further, we demonstrate LPPO II have low propensity for resistance development, likely due to their rapid membrane-targeting mode of action. Finally, we reveal that LPPO II are not toxic to either eukaryotic cells or model animals when administered orally or topically. Collectively, these results suggest that LPPO II are highly promising compounds for development into pharmaceuticals.
... Several years ago, we developed promising antibacterial compounds termed lipophosphonoxins (LPPOs) exhibiting significant antibacterial activities against a wide range of bacteria, including multidrugresistant strains, with no cytotoxicity on human cells at bactericidal concentrations [13][14][15] . LPPOs act through permeabilization of the bacterial membrane leading to its disruption and cell death. ...
... The first LPPO generation 16 displayed significant antibacterial activities against Gram-positive pathogens. Further modifications led to the second LPPO generation with improved antibacterial activities against both Gram-positive and Gram-negative bacteria 15 . Consequently, selected second generation LPPOs have been successfully evaluated as additives to surgical bone cements to prevent infections 17 and as component of nanofiber dressing capable of reducing wound infection in mice 18 . ...
... The exact stoichiometry of the pores is not yet clear. It could resemble varying number of monomers in oligomeric pores, which was described for LPPO II 15 . These results are consistent with other, structurally related members of the studied generation of LEGO-LPPOs 31 . ...
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.
... In parallel, we have previously synthesized innovative compounds known as lipophosphonoxins (LPPO), which exhibit potent antibacterial activities without exhibiting toxic effects on human cells [14][15][16]. The antibacterial mechanism of action of LPPO involves cell membrane permeabilization, leading to cell death. ...
... Furthermore, second-generation LPPOs have shown promise as additives to bone cement with antimicrobial effects [17]. Unlike typical antimicrobial drugs, LPPOs offer a distinct mechanism of action preventing microbes from developing cross-resistance [16]. This concept has been validated through a series of in vitro and in vivo experiments [18]. ...
... In this study, we utilized the same LPPO molecule as described in our previously published work [18]. The second-generation LPPO, DR-6180, was synthesized in multigram quantities following the method outlined in an earlier report [16]. ...
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.
... Several years ago, we developed promising antibacterial compounds termed lipophosphonoxins (LPPOs) exhibiting signi cant antibacterial activities against a wide range of bacteria, including multidrugresistant strains, with no cytotoxicity on human cells at bactericidal concentrations 8- 10 . LPPOs act through permeabilization of the bacterial membrane leading to its disruption and cell death. ...
... The rst LPPO generation 11 displayed signi cant antibacterial activities against Gram-positive pathogens. Further modi cations led to the second LPPO generation with improved antibacterial activities against both Gram-positive and Gramnegative bacteria 10 . Consequently, selected second generation LPPOs have been successfully evaluated as additives to surgical bone cements to prevent infections 12 and as component of nano ber dressing capable of reducing wound infection in mice 13 . ...
... The exact stoichiometry of the pores is not yet clear. It could resemble varying number of monomers in oligomeric pores, which was described for LPPO II 10 . These results are consistent with other, structurally related members of the studied generation of LEGO-LPPOs 26 . ...
Finding effective antibiotics against multi-resistant strains of bacteria has been a challenging race. LEGO-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. Presence of the intact outer membrane had a greater impact on the LEGO-LPPO action than changes in the structure of the hydrophobic modules, considering that the reduced susceptibility of Gram-negatives was attributed to the presence of the outer membrane and its components. Using liposome leakage and in silico simulations, we showed that membranes with major content of phosphatidylethanolamine were more prone to LEGO-LPPO permeabilization.
... Here we present a study on a new series of antimicrobial compounds we have developed, called LEGO-LPPOs (Linker-evolved-group-optimized-Lipophosphonoxins) [4]. LEGO-LPPOs are synthetic antimicrobial peptidomimetics loosely based on original lipophopsphonoxins [5,6]. ...
... The first generation of lipophosphonoxins was active against Gram-positive bacteria [5,7], while the second generation was active against Gram-negative bacteria as well [6]. The third generation had undesired increased haemolytic activity. ...
... Critical micellar concentration (CMC) values were measured by capillary electrophoresis carried out in an Agilent 7100 CE instrument (Agilent Technologies, Waldbronn, Germany) as described in [6]. Hydrophobicity Index (CHIg) was measured by gradient chromatography (linear gradient HPLC method) and calculated, as described previously [4]. ...
Linker-Evolved-Group-Optimized-Lipophosphonoxins (LEGO-LPPO) are small synthetic modular peptidomimetics with promising antimicrobial activity. The LEGO-LPPO mechanism of antibacterial action has been determined to be the depolarization and disruption of bacterial membranes. Their modular nature is advantageous for fine tuning their biological properties. In order to optimize the structure of LEGO-LPPO even further, it is important to understand the interaction of LEGO-LPPO with bacterial membranes at the molecular level. In this work, we present the synthesis of five LEGO-LPPO (designated as 1_naph2-4-G to 5_naph2-4-G) molecules bearing fluorescent naphtylethyl moieties and their usage in the study of LEGO-LPPO behaviour in the membrane. Our goal was to characterize fluorescently labelled LEGO-LPPO under conditions that do not completely disrupt the membrane, mostly in the form of membrane-bound monomers. We observed the intramolecular interactions of hydrophobic modules of 1_naph2-4-G in the buffer by detecting dynamic naphthyl excimers and their disappearance after 1_naph2-4-G bind into the membranes. In the membrane, the molecule 1_naph2-4-G slightly affects the membrane fluidity of DOPG membranes above the phase transition. The naphthyl fluorophore itself has fast and almost unrestricted rotation around ethylene linking groups (rinf = 0.010), which indicates a considerable chaotropic effect of the hydrophobic modules of 1_naph2-4-G at the given depth of the membrane. 1_naph2-4-G proved to be a useful model for observing the interaction of LEGO-LPPO antibiotics with the phospholipid bilayer enabling us to decipher its effects on membrane state and dynamics; its binding and penetration into the membrane, its structure and the particular depth that it occupies.
... Such a re-design of the imino-sugar module increased the number of positive charges and thus increased the affinity to the cytoplasmic membrane of the Gram-negative bacteria (see Figure 3). The second-generation LPPOs demonstrated in vitro an increased efficacy against Gram-positive pathogens vs. first-generation (MIC < 1-6 mg/L) as well as antibacterial activity against Gram-negative pathogens, including clinically relevant strains of Escherichia coli, Pseudomonas aeruginosa, and Salmonella Enteritidis [22]. Both generations of LPPOs share the same mode of action-the creation of pores in the cytoplasmic membrane, resulting in the efflux of the bacterial cytosol and cell disintegration. ...
... As in the first-generation, second-generation showed no effect on the eukaryotic cells at their bactericidal concentrations, no in vivo inhibition of biosynthesis of the cell macromolecules (DNA, RNA, protein, peptidoglycan, and membrane lipids), excellent thermostability and stability in low pH, and inability to pass through the Caco-2 monolayer. The second-generation LPPOs demonstrated in vitro an increased efficacy against Gram-positive pathogens vs. first-generation (MIC < 1-6 mg/L) as well as antibacterial activity against Gram-negative pathogens, including clinically relevant strains of Escherichia coli, Pseudomonas aeruginosa, and Salmonella Enteritidis [22]. Both generations of LPPOs share the same mode of action-the creation of pores in the cytoplasmic membrane, resulting in the efflux of the bacterial cytosol and cell disintegration. ...
... The Maximum Tolerated Dose (MTD) was investigated in mice using an orally administered dose of 2000 mg/kg of body weight. No death, body weight loss, or gross pathology changes were observed with either compound during a period of two weeks [22]. Skin irritation test was performed in rabbits and lasted for at least one week. ...
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.
... Lipophosphonoxins (LPPO) are novel compounds synthesized and patented by Rejman D. et al. in 2012 [89]. More recently, the same author and other group of investigators [90], using the first generation of LPPO, came up with a novel second generation of LPPO derivatives (LPPO II) by changing their building modules thus enhancing their biological activity. Of all the derivatives, compound 2p (Fig. 11) was the most active in most of the bacterial strains used ( Table 2). ...
... Of all the derivatives, compound 2p (Fig. 11) was the most active in most of the bacterial strains used ( Table 2). Further results allowed identifying the mechanism behind the compound's antibacterial activity which was through a cytoplasmic membrane disrupting mechanism in Gram-positive and Gram-negative bacteria [90]. ...
... The antibacterial effect is similar for the two classes of compounds and is mediated mainly by interference with the bacteria's cell membrane, ultimately leading to cell death. This interference can be triggered by cytoplasmic membrane disruption [90,92,94,97] by altering the bacteria's outer membrane permeability [97] or, for example, by targeting the Table 14 Structure, disease, and the pharmaceutical company of the guanidine-bearing drugs in the market for the treatment of protozoal infections. phospholipids through a synergy between the lactone ring and the polar head of the phospholipid and its guanidyl side-chain [148]. ...
Guanidines are fascinating small nitrogen-rich organic compounds, which have been frequently associated with a wide range of biological activities. This is mainly due to their interesting chemical features. For these reasons, for the past decades, researchers have been synthesizing and evaluating guanidine derivatives. In fact, there are currently on the market several guanidine-bearing drugs. Given the broad panoply of pharmacological activities displayed by guanidine compounds, in this review, we chose to focus on antitumor, antibacterial, antiviral, antifungal, and antiprotozoal activities presented by several natural and synthetic guanidine derivatives, which are undergoing preclinical and clinical studies from January 2010 to January 2023. Moreover, we also present guanidine-containing drugs currently in the market for the treatment of cancer and several infectious diseases. In the preclinical and clinical setting, most of the synthesized and natural guanidine derivatives are being evaluated as antitumor and antibacterial agents. Even though DNA is the most known target of this type of compounds, their cytotoxicity also involves several other different mechanisms, such as interference with bacterial cell membranes, reactive oxygen species (ROS) formation, mitochondrial-mediated apoptosis, mediated-Rac1 inhibition, among others. As for the compounds already used as pharmacological drugs, their main application is in the treatment of different types of cancer, such as breast, lung, prostate, and leukemia. Guanidine-containing drugs are also being used for the treatment of bacterial, antiprotozoal, antiviral infections and, recently, have been proposed for the treatment of COVID-19. To conclude, the guanidine group is a privileged scaffold in drug design. Its remarkable cytotoxic activities, especially in the field of oncology, still make it suitable for a deeper investigation to afford more efficient and target-specific drugs.
... By redesigning the iminosugar module so that it bears more positive charges, we developed the second generation of LPPOs (LPPO II) with increased efficacy (MIC <1−6 mg/L) against Gram-positive species and an extended antibacterial activity range that now also includes serious Gramnegative pathogens such as clinically relevant strains of Escherichia coli, Pseudomonas aeruginosa, and Salmonella Enteritidis. 19 LPPO II cause serious damage to the bacterial cell membrane, efflux of the bacterial cytosol, and cell disintegration. 20 Employing model membranes (liposomes and black lipid membranes), we demonstrated that LPPO II act by creating pores in the membrane. ...
... In LPPO III, NM was replaced with various simple ester groups to obtain a series of new derivatives 10a−o (Table 1) employing the same chemistry (Scheme 1) as in our original study. 19 Diethyl (4) or dimethyl vinylphosphonate (6) served as starting material. R 1 and R 2 groups were subsequently installed by reaction of monoethyl (5) or monomethyl vinylphosphonate with the appropriate hydroxyl derivative R 1 OH and R 2 OH either using TPSCl (2,4,6-triisopropylbenzenesulfonylchloride) as condensing agent or via phosphonochloridate generated from monomethyl vinylphosphonate with oxalylchloride/DMF. ...
... Importantly, antibacterial activities of LEGO-LPPOs are virtually unaffected by serum albumins, unlike their predecessors, LPPO II. 19 Finally, in vitro and in vivo skin and eye irritability tests with selected LEGO-LPPOs proved their safety for potential topical applications. Furthermore, oral and subcutaneous administration of 25 demonstrated its potential for systemic use. ...
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.
... The minimum inhibitory concentration values of the best inhibitors were in the 1-12 mg/L range, while their cytotoxic concentrations against human cell lines were significantly above this range [17][18][19]. Based on a study of the mechanism of action for the first generation, the second generation of LPPOs was designed and synthesized, providing compounds with a broadened spectrum of activity against several clinically relevant Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) [18,20]. Currently, the third and fourth generations of LPPOs are under development. ...
... They seem to outperform both first-and second-generation LPPOs in terms of antibacterial activity and selectivity. LPPOs of the first, second, and third generations do not irritate skin, and since they are not absorbed into the GIT, they are not toxic at peroral application (MTD for p.o. administration in mice of second generation LPPO is up to 2000 mg/kg of body weight) [20], which makes them ideal for use in the treatment of endodontic infections. ...
... Compound DR-6328 was prepared according to published procedure [20]. The compound is coded as 8b in this article. ...
(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.
... 13,15 One of the SAMPs with signicant antimicrobial activities are compounds termed lipophosphonoxins (LPPO) which we have designed and synthesized. LPPOs are synthetic small molecules that exhibit considerable antibacterial activity against a broad spectrum of bacteria, including multidrugresistant strains, without cytotoxicity on human cells at bactericidal concentrations [17][18][19] and low propensity to develop bacterial resistance. Our studies have demonstrated that LPPOs act by permeabilizing the bacterial membrane, leading to its disruption and cell death. ...
... In this generation, the PM is a hydrophilic moiety with an increased positive charge. 19,20 LPPO II has already been successfully evaluated as an antibacterial additive in the bone cement 21 and as a wound dressings based on the polycaprolactone nanober scaffold (NANO) releasing second generation lipophosphonoxin (LPPO) as the antibacterial agent. 22 Major structural alteration led to the newest generation called LEGO-LPPO, 23 which demonstrated activity against both G + and G − bacteria, and improved selectivity. ...
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.
... 17 (B) pK a of protons α to functional groups found in conjugate addition electrophiles. 19 Perzinfotel which has been investigated as an anti-stroke agent, 24 antibacterial agents, 25 and as enzyme inhibitors 26 (Fig. 2). Existing syntheses of these compounds include: Arbuzov reactions between phosphites and β-aminoalkyl halides 27,28 and nucleophilic substitution between β-halophosphonates and amines. ...
... Examples of biologically active β-aminophosph(in/on)ates.[24][25][26] ...
Thiourea catalysts activated α,β-unsaturated phosphonates and phosphinates toward conjugate addition by amines to give β-aminophosphonates and β-aminophosphinates. The organocatalytic methodology was used to synthesise 15 β-aminophosphonates and -phosphinates in yields up to 99%. A gram-scale example furnished the corresponding β-aminophosphonate in an isolated yield of 99% with 97% catalyst recovery. Based on mechanistic experiments, hydrogen bonding between the phosphoryl oxygen and thiourea are proposed to play a crucial role in substrate activation.
... PCL-based nanofibrous wound dressings were prepared by blending electrospinning technology 17,18 , with the addition of increasing concentrations (0, 2, 5, and 10 wt%) of LPPO DR-6180 (hereafter LPPO). LPPO was selected for its strong antibacterial properties and low cytotoxicity and was synthesized according to the procedure published previously 6 . The quality of the prepared nanofibrous materials was assessed by scanning electron microscopy (SEM). ...
... As shown in Tables S2 and S3, the amounts of LPPO detected in blood and liver correlated with its amounts in the NANO-LPPO composite. However, the detected systemic LPPO levels were negligible from the cytotoxicity point of view 6 . ...
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.
... LPPO. The second-generation LPPO DR-6180 ( Figure 1) was synthesized in multigram quantities according to the procedure reported elsewhere (11). ...
Background/Aim
This study investigated the therapeutic potential of lipophosphonoxin (LPPO), an antibacterial agent, loaded into polycaprolactone nanofiber dressings (NANO-LPPO) for full-thickness wound healing. Using a porcine model, we aimed to assess the impact of areal weight of the dressing (10, 20 and 30 g/m²) on wound-healing outcomes and validate findings from previous murine studies.
Materials and Methods
Full-thickness wounds were created on porcine skin and treated with the NANO-LPPO dressings of differing thickness. Positive control (Aquacel Ag+) and standard control (Jelonet) groups were included for comparison. Wound-healing progression was evaluated macroscopically and on the histological level.
Results
Macroscopic observations indicated no signs of infection in any group, with wounds covered by scabs by day 14. Thicker dressings (areal weights of 30 and 20 g/m²) demonstrated superior performance in promoting the formation of granulation tissue and healing compared to the thinner version (areal weight of 10 g/m²). LPPO-loading enhanced scaffold wettability and biodegradability without impairing healing outcomes. Both control groups exhibited similar healing characteristics.
Conclusion
The findings underscore the importance of optimizing dressing thickness for effective wound healing. NANO-LPPO dressings exhibit translational potential as a therapeutic option for full-thickness wounds, warranting further preclinical and regulatory evaluation to support clinical application.
... LPPO (Fig. 3) are synthetic small molecules that exhibit significant antibacterial activity against a broad spectrum of bacteria, including multidrugresistant strains, without cytotoxicity on human cells at bactericidal concentrations. [89][90][91] The antibacterial mechanism of LPPO involves permeabilization of the bacterial membrane, resulting in disruption and cell death. Thus, we have developed a new concept of wound dressing, based on the NANO that releases second-generation LPPO as an antibacterial agent. ...
... Black lipid membrane measurements. Planar lipid membrane experiments were performed as described previously 92 . The electrolyte solution contained 1 M KCl and 10 mM Tris-HCl (pH 7.4). ...
The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.
... The minimum inhibitory concentration (MIC) values were in the 1−12 mg/L range, while their cytotoxic concentrations against human cell lines were above this range (IC 50 60−100 mg/L). 8,9 However, LPPO I compounds are ineffective against Gramnegative bacteria. By redesigning the iminosugar module, so that it bore more positive charges, we developed the second generation of LPPOs (LPPOs II) with increased efficacy (MIC < 1−6 mg/L) against Gram-positive species and an extended antibacterial activity range that now also includes serious Gram-negative pathogens, such as clinically relevant strains of Escherichia coli, Pseudomonas aeruginosa, and Salmonella enteritidis. ...
Successful surgeries involving orthopedic implants depend on the avoidance of biofilm development on the implant surface during the early postoperative period. Here, we investigate the potential of novel antibacterial compounds—second-generation lipophosphonoxins (LPPOs II)—as additives to surgical bone cements. We demonstrate (i) excellent thermostability of LPPOs II, which is essential to withstand elevated temperatures during exothermic cement polymerization; (ii) unchanged tensile strength and elongation at the break properties of the composite cements containing LPPOs II compared to cements without additives; (iii) convenient elution kinetics on the order of days; and (iv) the strong antibiofilm activity of the LPPO II-loaded cements even against bacteria resistant to the medicinally utilized antibiotic, gentamicin. Thus, LPPOs II display promising potential as antimicrobial additives to surgical bone cements.
... The ability of compounds 5 f and 5 h with good antibacterial activity against MRSA to penetrate the bacterial cell membrane was studied using a PI assay [23]. As can be seen from Fig. 4, both compounds were efficient in penetrating the membranes of S. aureus at concentrations of 4 μg ml −1 and 8 μg ml −1 and are more effective than the positive control melittin, a peptide which is a cytoplasmic membrane pore-forming toxin [26,27]. The compounds tested exhibit a concentrationdependent effect on the penetration of the bacterial cell membrane. ...
Methicillin-resistant Staphylococcus aureus (MRSA) infection is a major threat to human health due to its resistance to almost all classes of antibiotics. Discovery of novel antibacterial agents with new structures which combat the pathogens responsible for MRSA is urgent. In this study, three series of benzyl phenyl sulfide derivatives were designed and synthesized, and their antibacterial activity against eleven MRSA strains were evaluated. The results showed that two series of the synthetic compounds (5a-5l and 12p-12u) exhibit potent antibacterial activity against S. aureus and MRSA, with minimum inhibitory concentrations of 2–64 μg/mL. The structure-activity relationships are discussed and the mechanism of the antibacterial activity was shown to involve the destruction of the bacterial cell membrane. Finally, the MTT assay results suggest that the toxicity of compounds 5f and 5h is selective between bacteria and mammalian cells.
... Black lipid membrane measurements. Planar lipid membrane experiments were performed as described previously (62,63). ...
Colicin U is a protein produced by the bacterium Shigella boydii (serovars 1 and 8). It exerts antibacterial activity against strains of the enterobacterial genera Shigella and Escherichia. Here we report that colicin U forms voltage-dependent pores in planar lipid membranes; its single-pore conductance was found to be about 22 pS in 1M KCl, at pH 6 under 80 mV in asolectin bilayers. In agreement with the high degree of homology between their C-terminal domains, colicin U shares some pore characteristics with the related colicins A and B. Colicin U pores are strongly pH dependent, and as we deduced from the activity of colicin U in planar membranes at different protein concentrations, they have a monomeric pore structure. However, in contrast to related colicins, we observed a very low cationic selectivity of colicin U pores (1.5/1 – K ⁺ /Cl ⁻ at pH 6) along with their atypical voltage gating. Finally, using nonelectrolytes, we determined the inner diameter of the pores to be in the range of 0.7 – 1 nm, which is similar to colicin Ia, but with a considerably different inner profile.
Importance Currently, a dramatic increase in antibiotic resistance is driving researchers to find new antimicrobial agents. Large group of toxins called bacteriocins appears to be very promising from this point of view, especially because their narrow killing spectrum allows specific targeting against selected bacterial strains. Colicins are a subgroup of bacteriocins that act on Gram-negative bacteria. To date, some colicins are commercially used for the treatment of animals (1) and tested as a component of engineered species-specific antimicrobial peptides, studied for the potential treatment of humans (2). Here we present a thorough single-molecule study of colicin U which leads to a better understanding of its mode of action. It extends the range of characterized colicins available for possible future medical applications.
... Kovalchuk et al. (2007) showed that a complex of natural cytokines and AMPs (CCAP or Superlymph) inhibits virus proliferation in vitro. Therefore, due to their small molecular weight, good thermal stability and broad antimicrobial spectrum, AMPs are expected to replace antibiotics as new and highly effective antibacterial drugs (Presicce et al. 2009;Seydlová et al. 2017). ...
Antimicrobial peptides (AMPs) are cationic small peptide chains that have good antimicrobial activity against a variety of bacteria, fungi, and viruses. AMP-17 is a recombinant insect AMP obtained by a prokaryotic expression system. However, the full antifungal activity, physicochemical characteristics, and cytotoxicity of AMP-17 were previously unknown. AMP-17 was shown to have good antifungal activity against five pathogenic fungi, with minimum inhibitory concentrations (MIC) of 9.375–18.75 μg/ml, and minimum fungicidal concentrations (MFC) of 18.75–37.5 μg/ml. Notably, the antifungal activity of AMP-17 against Cryptococcus neoformans was superior to that of other Candida spp. In addition, the hemolytic rate of AMP-17 was only 1.47%, even at the high concentration of 16 × MIC. AMP-17 was insensitive to temperature and high salt ion concentration, with temperatures of 98°C and –80°C, and NaCl and MgCl2 concentrations of 50–200 mmol/l, having no significant effect on antifungal activity. However, AMP-17 was sensitive to proteases, trypsin, pepsin, and proteinase K. The elucidation of antifungal activity, physicochemical properties and cytotoxicity of AMP-17 provided an experimental basis for its safety evaluation and application, as well as indicated that AMP-17 might be a promising drug.
... The pore-forming activity of colicin Z (concentrations up to 340 nM) was tested using conductivity measurements in 1 M KCl, 10 mM HEPES, pH 6 on black lipid membranes composed of DPhPG, E. coli polar lipid extract (Avanti Polar Lipids, Alabaster, AL, USA), or soybean lipids (Asolectin-Type II, Merck KGaA, Darmstadt, Germany). Experiments were performed as described previously 50 . The effect of colZ on peptidoglycan was tested using the dye release method described in Zhou et al. 51 . ...
Colicin production in Escherichia coli (E. coli) strains represents an important trait with regard to microbial survival and competition in the complex intestinal environment. A novel colicin type, colicin Z (26.3 kDa), was described as a product of an original producer, extraintestinal E. coli B1356 strain, isolated from the anorectal abscess of a 17 years-old man. The 4,007 bp plasmid (pColZ) was completely sequenced and colicin Z activity (cza) and colicin Z immunity (czi) genes were identified. The cza and czi genes are transcribed in opposite directions and encode for 237 and 151 amino acid-long proteins, respectively. Colicin Z shows a narrow inhibitory spectrum, being active only against enteroinvasive E. coli (EIEC) and Shigella strains via CjrC receptor recognition and CjrB- and ExbB-, ExbD-mediated colicin translocation. All tested EIEC and Shigella strains isolated between the years 1958–2010 were sensitive to colicin Z. The lethal effect of colicin Z was found to be directed against cell wall peptidoglycan (PG) resulting in PG degradation, as revealed by experiments with Remazol Brilliant Blue-stained purified peptidoglycans and with MALDI-TOF MS analyses of treated PG. Colicin Z represents a new class of colicins that is structurally and functionally distinct from previously studied colicin types.
... Despite CPAs being an alternative for treating drug-resistant bacteria, their transformation into molecules of therapeutic use is a highly daunting task owing to several disadvantages, including their large size, hemolytic activity, proteolytic instability, poor salt resistance, and cytotoxicity 14 . Hence, amphiphilic small molecules containing a lipophilic groups and cationic groups exhibiting controlled hemolytic activity are highly imperative for the discovery of new antibiotics and to overcome peptide-associated disadvantages, including proteolytic instability and poor cell permeability 15,16 . Thus, small molecules with structural features of CPAs are considered as a prominent strategy that may help in the discovery of small-molecule antibacterials. ...
The emergence of multi-drug resistant bacteria forces the therapeutic world into a position, where the development of new and alternative kind of antibiotics is highly important. Herein, we report the development of triazine-based amphiphilic small molecular antibacterial agents as mimics of lysine- and arginine-based cationic peptide antibiotics (CPAs). These compounds were screened against a panel of both Gram-positive and Gram-negative bacterial strains. Further, anti-inflammatory evaluation of these compounds led to the identification of four efficient compounds, DG-5, DG-6, DL-5, and DL-6. These compounds displayed significant potency against drug-resistant bacteria, including methicillin-resistant S. aureus (MRSA), multidrug-resistant P. aeruginosa (MDRPA), and vancomycin-resistant E. faecium (VREF). Mechanistic studies, including cytoplasmic membrane depolarization, confocal imaging and flow cytometry suggest that DG-5, DG-6, and DL-5 kill bacteria by targeting bacterial membrane, while DL-6 follows intracellular targeting mechanism. We also demonstrate that these molecules have therapeutic potential by showing the efficiency of DG-5 in preventing the lung inflammation of lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. More interestingly, DL-6 exhibited impressive potency on atopic dermatitis (AD)-like skin lesions in BALB/c mice model by suppressing pro-inflammatory cytokines. Collectively, these results suggest that they can serve a new class of antimicrobial, anti-inflammatory and anti-atopic agents with promising therapeutic potential.
... The final samples were examined in an FEI Nova NanoSEM scanning electron microscope (FEI, Brno, Czech Republic) at 5 kV, using concentric backscatter (CBS) and through-the-lens (TLD) detectors. The cells were imaged according to a protocol described previously (59). ...
σ I from Bacillus subtilis is a σ factor associating with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here we provide a comprehensive characterization of this transcriptional regulator. By RNA-seq of wt and σ I -null strains at 37°C and 52°C we identified ∼130 genes affected by the absence of σ I . Further analysis revealed that the majority of these genes were affected by σ I indirectly. The σ I regulon, i.e., the genes directly regulated by σ I , consists of 16 genes of which eight (the dhb and yku operons) are involved in iron metabolism. The involvement of σ I in iron metabolism was confirmed phenotypically. Next, we set up an in vitro transcription system and defined and experimentally validated the promoter sequence logo that, in addition to -35 and -10 regions, also contains extended -35 and -10 motifs. Thus, σ I -dependent promoters are relatively information-rich in comparison with most other promoters. In summary, this study supplies information about the least explored σ factor from the industrially important model organism B. subtilis .
Importance In bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons ( i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σ I regulon from the industrially important model Gram-positive bacterium – Bacillus subtilis . We reveal that σ I affects expression of ∼ 130 genes, of which 16 are directly regulated by σ I , including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σ I -dependent transcription. This study thus provides a comprehensive view on this underexplored component of the B. subtilis transcription machinery.
Significance: Negative pressure wound therapy (NPWT) has been in practice for decades, proving its utility in many applications, ranging from acutely infected wounds to complex combat wounds and skin grafting. It has been routinely demonstrated that NPWT has superior wound healing outcomes compared with previous standard-of-care therapies. However, the technique involves some challenges related to each of the components that comprise the therapy. The purpose of this article is to highlight the challenges, introduce the recent advancements, and discuss about the future directions in NPWT systems. Recent Advances: New techniques and materials have been developed to improve the currently used NPWT systems with promising results when utilized with appropriate indications. Many advancements have been introduced in modes of negative pressure delivery, pumps, interface dressings, adhesive dressings, and tubing technology. Critical Issues: An optimal NPWT system would avoid the common problems such as failure to deliver negative pressure due to loss of an airtight seal or tissue ingrowth into the interface dressing causing painful dressing changes and bleeding. Other challenges include infection control and patient pain and discomfort that may contribute to noncompliance. Future Directions: Many studies have been performed to evaluate the optimal combination of settings and components in various wounds; however, there is still no clear "best" answer for many specific patient-wound scenarios. Novel and emerging tissue engineering and regenerative medicine approaches could potentially be utilized in the future NPWT systems and thus, this review will discuss some novel ideas for future considerations.
Infections caused by drug-resistant bacteria have emerged to be one of the greatest threats to global public health, and new antimicrobial agents with novel mechanisms of action hence are in an urgent need to combat bacterial resistance. Herein, we reported the design, synthesis, and antibacterial evaluation of novel honokiol derivatives as mimics of antimicrobial peptides (AMPs). These mimics showed potent antimicrobial properties against Gram-positive bacteria. Among them, the most promising compound 13b exhibited excellent antibacterial activity, rapid bactericidal properties, avoidance of antibiotic resistance, and weak hemolytic and cytotoxic activities. In addition, compound 13b not only inhibited the biofilm formation but also destroy the preformed biofilm. Mechanism studies further revealed that compound 13b killed bacteria rapidly by interrupting the bacterial membrane. More intriguingly, compound 13b exhibited potent in vivo antibacterial efficacy in a mouse septicemia model induced by Staphylococcus aureus ATCC43300. These results highlight the potential of 13b to be used as therapeutic agents.
When a monosaccharide (or sugar fragment of any size) is condensed with either an aliphatic or aromatic alcohol, or another sugar moiety through an oxygen, a glycoside bond is formed. General examples of O-glycosides are shown in Scheme 2.1.
Conventional Nuclear Magnetic Resonance (NMR) analysis relies on H-H/C-H interactions. However, these interactions are sometimes insufficient for an accurate and precise NMR analysis. In this study, we show that ³¹P NMR parameters can provide critical structural insights into the stereochemistry of phosphorus-containing compounds. For this purpose, we prepared a set of model phosphorus-based proline derivatives, separated diastereoisomers, and determined their absolute configuration by single-crystal X-ray diffraction. After supplementing these results by electronic circular dichroism (ECD) spectroscopy, we combined experimental data and DFT calculations from our model compounds to perform a detailed conformational analysis, thereby determining their relative configuration. Overall, our findings establish an experimental paradigm for combining ³¹P NMR spectroscopy with other optical methods to facilitate the stereochemical analysis of phosphorus-containing compounds.
The Staphylococcus aureus can switch to a transient genotype-invariant dormancy, known as a persister, to survive treatment with high doses of antibiotics. This transient persister is an important reason underlying its resistance. There is an urgent need to find new antibacterial agents capable of eradicating methicillin-resistant S. aureus (MRSA) persisters. In this study, 37 new derivatives of cajaninstilbene acid (CSA) were designed and synthesized, and their biological activity against MRSA persisters was evaluated. Most of the newly synthesized derivatives exhibit more potent antimicrobial properties against S. aureus and MRSA than CSA itself, and 23 of the 37 derivatives show a tendency to eradicate MRSA persisters. A representative compound (A6) was demonstrated to target bacterial cell membranes. It eradicated the adherent biofilm of MRSA in a concentration dependent manner, and showed a synergistic antibacterial effect with piperacilin. In a model mouse abscess caused by MRSA persisters, A6 effectively reduced the bacterial load in vivo. These results indicate that A6 is a potential candidate for treatment of MRSA persister infections.
Lipophosphonoxins (LPPOs) are small modular synthetic antibacterial compounds that target the cytoplasmic membrane. First-generation LPPOs (LPPO I) exhibit an antimicrobial activity against Gram-positive bacteria; however they do not exhibit any activity against Gram-negatives. Second-generation LPPOs (LPPO II) also exhibit broadened activity against Gram-negatives. We investigated the reasons behind this different susceptibility of bacteria to the two generations of LPPOs using model membranes and the living model bacteria Bacillus subtilis and Escherichia coli . We show that both generations of LPPOs form oligomeric conductive pores and permeabilize the bacterial membrane of sensitive cells. LPPO activity is not affected by the value of the target membrane potential, and thus they are also active against persister cells. The insensitivity of Gram-negative bacteria to LPPO I is probably caused by the barrier function of the outer membrane with LPS. LPPO I is almost incapable of overcoming the outer membrane in living cells, and the presence of LPS in liposomes substantially reduces their activity. Further, the antimicrobial activity of LPPO is also influenced by the phospholipid composition of the target membrane. A higher proportion of phospholipids with neutral charge such as phosphatidylethanolamine or phosphatidylcholine reduces the LPPO permeabilizing potential.
Covering: 2017–2019
Guanidine natural products isolated from microorganisms, marine invertebrates and terrestrial plants, amphibians and spiders, represented by non-ribosomal peptides, guanidine-bearing polyketides, alkaloids, terpenoids and shikimic acid derived, are the subject of this review. The topics include the discovery of new metabolites, total synthesis of natural guanidine compounds, biological activity and mechanism-of-action, biosynthesis and ecological functions.
Antimicrobial peptides (AMP) with anticancer activity have drawn remarkable attention in modern treatments. However, long peptide length and protease instability are the most addressing factors, which hampers their further development as therapeutic agents. In view of this, herein, we designed and synthesized a series of AZT-based cationic small molecule incorporating a variety of hydrophobic groups and cationic charges, including amine and guanidine groups to mimic the amphipathic structure of AMPs. These compounds were evaluated for their antibacterial activity against Gram-positive and Gram-negative bacteria. Through an extensive structure activity relationship study (SAR), we identified ADG-2e as the most potent antibacterial agent, which exhibited remarkable potency against drug resistant bacterial strains such as MRSA and MDRPA. Further, ADG-2e was examined for their anti-metastatic ability by investigating the cancer cell migration and invasiveness through scratch wound-healing assay and transwell invasive assay, respectively. In addition, time-lapse cell tracking analysis also performed for analyzing the cell movement pattern. Treatment of ADG-2e against metastatic breast cancer cells (MDA-MB-231) suppressed tumor cell migration by multi-directional lamellipodium formation, indicating their anti-metastatic potential. Thus, our cationic AZT based small molecules may evolve as an appealing class of antibacterial agents with anti-metastasis potential.
Antibiotic resistance is one of the biggest threats to public health, and new antibacterial agents hence are in an urgent need to combat infectious diseases caused by multidrug-resistant (MDR) pathogens. Utilizing dimerization strategy, we rationally designed and efficiently synthesized a new series of small molecules-dimeric lysine alkylamides as mimics of AMPs. Evaluation of these mimics against a panel of Gram-positive and Gram-negative bacteria including MDR strains were performed, and a broad-spectrum and potent compound 3d was identified. This compound displayed high specificity toward bacteria over mammalian cell. Time−kill kinetics and mechanistic studies suggest that compound 3d quickly eliminated bacteria in a bactericidal mode by disrupting bacterial cell membrane. In addition, lead compound 3d could inhibit biofilm formation and did not develop drug resistance in S.aureus and E.coli over 14 passages. These results suggested that dimeric lysine nonylamide has immense potential as a new type of novel small molecular agent to combat antibiotic resistance.
Inflammatory bowel disease is thought to be caused by an aberrant immune response to gut bacteria in a genetically susceptible host. The gut microbiota plays an important role in the pathogenesis and complications of the two main inflammatory bowel diseases: Crohn's disease (CD) and ulcerative colitis. Alterations in gut microbiota, and specifically reduced intestinal microbial diversity, have been found to be associated with chronic gut inflammation in these disorders. Specific bacterial pathogens, such as virulent Escherichia coli strains, Bacteroides spp, and Mycobacterium avium subspecies paratuberculosis, have been linked to the pathogenesis of inflammatory bowel disease. Antibiotics may influence the course of these diseases by decreasing concentrations of bacteria in the gut lumen and altering the composition of intestinal microbiota. Different antibiotics, including ciprofloxacin, metronidazole, the combination of both, rifaximin, and anti-tuberculous regimens have been evaluated in clinical trials for the treatment of inflammatory bowel disease. For the treatment of active luminal CD, antibiotics may have a modest effect in decreasing disease activity and achieving remission, and are more effective in patients with disease involving the colon. Rifamixin, a non absorbable rifamycin has shown promising results. Treatment of suppurative complications of CD such as abscesses and fistulas, includes drainage and antibiotic therapy, most often ciprofloxacin, metronidazole, or a combination of both. Antibiotics might also play a role in maintenance of remission and prevention of post operative recurrence of CD. Data is more sparse for ulcerative colitis, and mostly consists of small trials evaluating ciprofloxacin, metronidazole and rifaximin. Most trials did not show a benefit for the treatment of active ulcerative colitis with antibiotics, though 2 meta-analyses concluded that antibiotic therapy is associated with a modest improvement in clinical symptoms. Antibiotics show a clinical benefit when used for the treatment of pouchitis. The downsides of antibiotic treatment, especially with recurrent or prolonged courses such as used in inflammatory bowel disease, are significant side effects that often cause intolerance to treatment, Clostridium dificile infection, and increasing antibiotic resistance. More studies are needed to define the exact role of antibiotics in inflammatory bowel diseases.
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.
Natural and synthetic membrane active antibacterial agents offer hope as potential solutions to the problem of bacterial resistance as the membrane-active nature imparts low propensity for the development of resistance. In this report norspermidine based antibacterial molecules were developed which displayed excellent antibacterial activity against various wild-type bacteria (Gram-positive and Gram-negative) and drug-resistant bacteria (methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium and β-lactam-resistant Klebsiella pneumoniae). In a novel structure-activity relationship study it has been shown how incorporation of an aromatic amino acid drastically improves selective antibacterial activity. Additionally, the effect of stereochemistry on activity, toxicity and plasma stability has also been studied. These rapidly bactericidal, membrane active antibacterial compounds do not trigger development of resistance in bacteria and hence bear immense potential as therapeutic agents to tackle multi-drug resistant bacterial infections.
Daptomycin is a lipopeptide antibiotic that is used clinically to treat severe infections caused by Gram-positive bacteria. Its bactericidal action involves the calcium-dependent binding to membranes containing phosphatidylglycerol, followed by the formation of membrane-associated oligomers. Bacterial cells exposed to daptomycin undergo membrane depolarization, suggesting the formation of channels or pores in the target membranes. We here used a liposome model to detect and characterize the permeability properties of the daptomycin pores. The pores are selective for cations, with permeabilities being highest for Na(+), K(+), and other alkali metal ions. The permeability is approximately twice lower for Mg(++), and lower again for the organic cations choline and hexamethonium. Anions are excluded, as is the zwitterion cysteine. These observations account for the observed depolarization of bacterial cells by daptomycin and suggest that under typical in vivo conditions depolarization is mainly due to sodium influx.
Antistaphylococcal agents commonly lack activity against Gram-negative bacteria like Escherichia coli owing to the permeability barrier presented by the outer membrane and/or the action of efflux transporters. When these intrinsic
resistance mechanisms are artificially compromised, such agents almost invariably demonstrate antibacterial activity against
Gram negatives. Here we show that this is not the case for the antibiotic daptomycin, whose target appears to be absent from
E. coli and other Gram-negative pathogens.
Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which
has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively
mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors,
accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed
description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription
initiation by alternative sigma factors and from imperfect control of transcription termination.
Small arylamide foldamers designed to mimic the amphiphilic nature of antimicrobial peptides (AMPs) have shown potent bactericidal
activity against both Gram-negative and Gram-positive strains without many of the drawbacks of natural AMPs. These foldamers
were shown to cause large changes in the permeability of the outer membrane of Escherichia coli. They cause more limited permeabilization of the inner membrane which reaches critical levels corresponding with the time
required to bring about bacterial cell death. Transcriptional profiling of E. coli treated with sublethal concentrations of the arylamides showed induction of genes related to membrane and oxidative stresses,
with some overlap with the effects observed for polymyxin B. Protein secretion into the periplasm and the outer membrane is
also compromised, possibly contributing to the lethality of the arylamide compounds. The induction of membrane stress response
regulons such as rcs coupled with morphological changes at the membrane observed by electron microscopy suggests that the activity of the arylamides
at the membrane represents a significant contribution to their mechanism of action.
The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.
A new method for accurately determining effective mobilities and electroosmotic flow rates for capillary electrophoresis is described. The proposed method can be performed using most commercial capillary electrophoresis instruments. Problems inherent to the conventional mobility determination method such as a variable electroosmotic flow during the run and migration through unthermostated regions of the capillary are eliminated with the use of the proposed method. In addition, very low effective mobilities and electroosmotic flow rates can be measured quickly and reproducibly. Also, cation mobilities and anion mobilities can be measured in a single run regardless of the magnitude or direction of the electroosmotic flow.
Although the influence of protein binding (PB) on antibacterial activity has been reported for many antibiotics and over many
years, there is currently no standardization for pharmacodynamic models that account for the impact of protein binding of
antimicrobial agents in vitro. This might explain the somewhat contradictory results obtained from different studies. Simple in vitro models which compare the MIC obtained in protein-free standard medium versus a protein-rich medium are prone to methodological
pitfalls and may lead to flawed conclusions. Within in vitro test systems, a range of test conditions, including source of protein, concentration of the tested antibiotic, temperature,
pH, electrolytes, and supplements may influence the impact of protein binding. As new antibiotics with a high degree of protein
binding are in clinical development, attention and action directed toward the optimization and standardization of testing
the impact of protein binding on the activity of antibiotics in vitro become even more urgent. In addition, the quantitative relationship between the effects of protein binding in vitro and in vivo needs to be established, since the physiological conditions differ. General recommendations for testing the impact of protein
binding in vitro are suggested.
Persistent infections involving slow-growing or non-growing bacteria are hard to treat with antibiotics that target biosynthetic processes in growing cells. Consequently, there is a need for antimicrobials that can treat infections containing dormant bacteria. In this Review, we discuss the emerging concept that disrupting the bacterial membrane bilayer or proteins that are integral to membrane function (including membrane potential and energy metabolism) in dormant bacteria is a strategy for treating persistent infections. The clinical applicability of these approaches is exemplified by the efficacy of lipoglycopeptides that damage bacterial membranes and of the diarylquinoline TMC207, which inhibits membrane-bound ATP synthase. Despite some drawbacks, membrane-active agents form an important new means of eradicating recalcitrant, non-growing bacteria.
In August 2000, the US Food and Drug Administration (FDA) approved ciprofloxacin hydrochloride (Cipro; Bayer) for management
of postexposure inhalational anthrax. This was the first antimicrobial drug approved by the FDA for use in treating an infection
due to a biological agent used intentionally. The terrorist attacks of 2001 involving anthrax underscore the imperative that
safe and effective drugs to manage such infections be readily available in the United States. The approval of ciprofloxacin
hydrochloride, which was made on the basis of a surrogate human marker of efficacy, made extensive use of data from an animal
model of disease. This represents a new direction in the development of efficacy data in support of drug approval and facilitates
the availability of those drugs for which there is an urgent need. This article presents the scientific data and regulatory
mechanism that supported the approval of ciprofloxacin hydrochloride for management of postexposure of inhalational anthrax.
Antimicrobial peptides (AMPs) are an essential part of innate immunity that evolved in most living organisms over 2.6 billion years to combat microbial challenge. These small cationic peptides are multifunctional as effectors of innate immunity on skin and mucosal surfaces and have demonstrated direct antimicrobial activity against various bacteria, viruses, fungi, and parasites. This review summarizes their progress to date as commercial antimicrobial drugs for topical and systemic indications.
Literature review.
Despite numerous clinical trials, no modified AMP has obtained Food & Drug Administration approval yet for any topical or systemic medical indications.
While AMPs are recognized as essential components of natural host innate immunity against microbial challenge, their usefulness as a new class of antimicrobial drugs still remains to be proven.
Novel cationic antimicrobial peptides typified by structures such as KKKKKKAAXAAWAAXAA-NH2, where X = Phe/Trp, and several of their analogues display high activity against a variety of bacteria but exhibit no hemolytic activity even at high dose levels in mammalian erythrocytes. To elucidate their mechanism of action and source of selectivity for bacterial membranes, phospholipid mixtures mimicking the compositions of natural bacterial membranes (containing anionic lipids) and mammalian membranes (containing zwitterionic lipids + cholesterol) were challenged with the peptides. We found that peptides readily inserted into bacterial lipid mixtures, although no insertion was detected in model "mammalian" membranes. The depth of peptide insertion into model bacterial membranes was estimated by Trp fluorescence quenching using doxyl groups variably positioned along the phospholipid acyl chains. Peptide antimicrobial activity generally increased with increasing depth of peptide insertion. The overall results, in conjunction with molecular modeling, support an initial electrostatic interaction step in which bacterial membranes attract and bind peptide dimers onto the bacterial surface, followed by the "sinking" of the hydrophobic core segment to a peptide sequence-dependent depth of approximately 2.5-8 A into the membrane, largely parallel to the membrane surface. Antimicrobial activity was likely enhanced by the fact that the peptide sequences contain AXXXA sequence motifs, which promote their dimerization, and possibly higher oligomerization, as assessed by SDS-polyacrylamide gel analysis and fluorescence resonance energy transfer experiments. The high selectivity of these peptides for nonmammalian membranes, combined with their activity toward a wide spectrum of Gram-negative and Gram-positive bacteria and yeast, while retaining water solubility, represent significant advantages of this class of peptides.
To use rodent models effectively in in-vivo investigations on oral drug and vaccine delivery, the conditions in the gastrointestinal tract must be understood. Some fundamental information is currently unavailable or incomplete. We have investigated the pH, water content and lymphoid tissue distribution along the gastrointestinal tract, as well as the stomach volume, as these were critical to our investigations on pH-responsive drug delivery and colonic vaccination. The observed values were compared with those in man as an indication of the validity of the rodent model. The mouse stomach pH was 3.0 (fed) and 4.0 (fasted), and the corresponding values in the rat were 3.2 (fed) and 3.9 (fasted). The mean intestinal pH was lower than that in man (<pH 5.2 in the mouse; <pH 6.6 in the rat). This brings into question the use of rodents in investigations on enteric-coated drug carriers targeted to the large intestine/distal gut. The water content in the gastrointestinal tract in the fed and fasted mouse was 0.98+/-0.4 and 0.81+/-1.3 mL, respectively, and in the fed and fasted rat was 7.8+/-1.5 and 3.2+/-1.8 mL. When normalized for body weight, there was more water per kg body weight in the gastrointestinal tracts of the mouse and rat, than in man. The stomach capacity was found to be approximately 0.4 and 3.4 mL for mice and rats, respectively. The low fluid volume and stomach capacity have implications for the testing of solid dosage forms in these animal models. Substantial amounts of lymphoid tissue analogous to small intestinal Peyer's patches were measured in the rat and mouse colon, showing the feasibility of colonic vaccination, a route which might prove to have different applications to the more commonly studied oral vaccines. The existence of lymphoid tissue in the mouse and rat caecum has also been reported.
Background:
The gastrointestinal tract is not the common infection site of Pseudomonas aeruginosa. The role of P. aeruginosa as a causative agent for diarrhea in children without pre-existing disease is controversial.
Methods:
From 2003 to 2012 we reviewed the records of 259 diarrheal patients less than 5 years of age whose stool culture grew P. aeruginosa. Virulence phenotypes of bacterial isolates were determined in vitro, including cytotoxicity, penetration and adherence to epithelial cells.
Results:
The presence of P. aeruginosa in children with diarrhea less than 5 years old is 0.91%. P. aeruginosa-associated diarrheal diseases were classified into 4 groups: Shanghai fever (enteric infection and sepsis) (5%), P. aeruginosa enterocolitis (15%), P. aeruginosa-related diarrhea (19%) and antibiotic-associated diarrhea (43%). The remaining patients had co-infection with other pathogens (18%). Shanghai fever was the most severe enteric disease with invasive infection and complications. The clinical features of P. aeruginosa enterocolitis were prolonged fever with bloody or mucoid diarrhea mimicking bacterial enterocolitis. The clinical features of P. aeruginosa-related diarrhea and antibiotic-associated diarrhea were similar to viral or toxin-mediated diarrhea. Compared with other P. aeruginosa-associated diarrheal diseases, patients with Shanghai fever were younger, usually infants, and the characteristic laboratory findings included leukopenia, thrombocytopenia, high C-reactive protein, hyponatremia and hyperglycemia. Except for Shanghai fever, antibiotic treatment is not recommended. Isolates from Shanghai fever were more cytotoxic and adherent than isolates from uncomplicated diarrheal patients.
Conclusions:
P. aeruginosa could be an enteric pathogen even in healthy children. Young age and highly virulent bacterial strains were risk factors for Shanghai fever.
Purpose of review:
Methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health issue worldwide over the last years. MRSA is frequently implicated in the development of skin and soft tissue infections, leading to significant increases in morbidity, mortality and overall healthcare costs.
Recent findings:
In order to face the threat of MRSA, major changes in clinical management of skin and soft tissue infections are required. The identification of populations at risk for the acquisition of infections due to MRSA, together with the improvement of the diagnostic techniques, is paramount. Moreover, a number of new antimicrobials with activity against MRSA have been recently developed and approved for the treatment of skin and soft tissue infections, however, the use of the new drugs in the wide clinical practice remains limited.
Summary:
We reviewed the current epidemiology of MRSA in skin and soft tissue infections, with particular focus on implications for clinical management. The potential role of new antibiotic options against MRSA infections is also discussed.
BACKGROUND
The escalating crisis of multidrug resistance is raising fears of untreatable infections caused by bacterial “superbugs.” However, many patients already suffer from infections that are effectively untreatable due to innate bacterial mechanisms for persistence. This phenomenon is caused by the formation of specialized persister cells that evade antibiotic killing and other stresses by entering a physiologically dormant state, irrespective of whether they possess genes enabling antibiotic resistance. The recalcitrance of persister cells is a major cause of prolonged and recurrent courses of infection that can eventually lead to complete antibiotic treatment failure. Regularly growing bacteria differentiate into persister cells stochastically at a basal rate, but this phenotypic conversion can also be induced by environmental cues indicative of imminent threats for the bacteria. Size and composition of the persister subpopulation in bacterial communities are largely controlled by stress signaling pathways, such as the general stress response or the SOS response, in conjunction with the second messenger (p)ppGpp that is almost always involved in persister formation. Consequently, persister formation is stimulated under conditions that favor the activation of these signaling pathways. Such conditions include bacterial biofilms and hostile host environments, as well as response to damage caused by sublethal concentrations of antibiotics.
ADVANCES
The limited comprehensive understanding of persister formation and survival is a critical issue in controlling persistent infections. However, recent work in the field has uncovered the molecular architecture of several cellular pathways underlying bacterial persistence, as well as the functional interactions that generate heterogeneous populations of persister cells. These results confirm the long-standing notion that persistence is intimately connected to slow growth or dormancy in the sense that a certain level of physiological quiescence is attained. Most prominently, the central role of toxin-antitoxin (TA) modules has been explained in considerable detail. In the model organism Escherichia coli K-12, two major pathways of persister formation via TA modules are both controlled by (p)ppGpp and involve toxin HokB and a panel of mRNA endonuclease toxins, respectively. Whereas activation of the membrane-associated toxin HokB depends on the enigmatic (GTPase) guanosine triphosphatase Obg and causes persister formation by abolishing the proton-motive force, mRNA endonuclease toxins are activated through antitoxin degradation by protease Lon and globally inhibit translation. In addition to these two pathways, toxin TisB is activated in response to DNA damage by the SOS response and promotes persister formation in a manner similar to HokB. Beyond TA modules, many additional factors (such as cellular energy metabolism or drug efflux) have been found to contribute to persister formation and survival, but their position in particular molecular pathways is often unclear. Altogether, this diversity of mechanisms drives the formation of a highly heterogeneous ensemble of persister cells that displays multistress and multidrug tolerance as the root of the recalcitrance of persistent infections.
OUTLOOK
Though recent advances in the field have greatly expanded our understanding of the molecular mechanisms underlying persister formation, important facets have remained elusive and should be addressed in future studies. One example is the upstream signaling input into the pathways mediating bacterial persister formation (e.g., the nature of the pacemaker driving stochastic persister formation). Similarly, it is often not well understood how—beyond the general idea of dormancy—persister cells can survive the action of lethal antibiotics. Finally, one curious aspect of the persister field is recurrent inconsistency between the results obtained by different groups. We speculate that these variations may be linked to subtle differences in experimental procedures inducing separate yet partially redundant pathways of persister formation. It is evident that the elucidation of this phenomenon may not only consolidate progress in the field but also offer the chance to gain insights into the molecular basis and control of bacterial persistence.
Bacterial persisters defy antibiotic treatment
Persister cells are phenotypic variants of regularly growing bacteria and survive lethal antibiotic treatment in a nongrowing, dormant state. Upon termination of treatment, the resuscitation of persister cells can replenish the population. Our Review focuses on the diverse molecular mechanisms that underlie bacterial persister formation and drive the heterogeneity of these cells. PMF, proton-motive force.
This review is the result of discussions that took place at the 5th MRSA Working Group Consensus Meeting and explores the possible treatment options available for different types of infections due to methicillin-resistant Staphylococcus aureus (MRSA), focusing on those antibiotics that could represent a valid alternative to vancomycin. In fact, whilst vancomycin remains a viable option, its therapy is moving towards individualised dosing. Other drugs, such as the new lipoglycopeptides (oritavancin, dalbavancin and telavancin) and third-generation cephalosporins (ceftaroline and ceftobiprole), are showing good in vitro potency and in vivo efficacy, especially for patients infected with micro-organisms with higher vancomycin minimum inhibitory concentrations (MICs). Tedizolid is an attractive agent for use both in hospital and community settings, but the post-marketing data will better clarify its potential. Daptomycin and linezolid have shown non-inferiority to vancomycin in the treatment of MRSA bacteraemia and non-inferiority/superiority to vancomycin in the treatment of hospital-acquired pneumonia. Thus, several options are available, but more data from clinical practice, especially for invasive infections, are needed to assign specific roles to each antibiotic and to definitely include them in the new antibacterial armamentarium.
Bacteria display an array of contact-dependent interaction systems that have evolved to facilitate direct cell-to-cell communication. We have previously identified a mode of bacterial communication mediated by nanotubes bridging neighboring cells. Here, we elucidate nanotube architecture, dynamics, and molecular components. Utilizing Bacillus subtilis as a model organism, we found that at low cell density, nanotubes exhibit remarkable complexity, existing as both intercellular tubes and extending tubes, with the latter frequently surrounding the cells in a “root-like” fashion. Observing nanotube formation in real time showed that these structures are formed in the course of minutes, displaying rapid movements. Utilizing a combination of super-resolution, light, and electron microscopy, we revealed that nanotubes are composed of chains of membranous segments harboring a continuous lumen. Furthermore, we discovered that a conserved calcineurin-like protein, YmdB, presents in nanotubes and is required for both nanotube production and intercellular molecular trade.
Antibiotic resistance is a critical global health care crisis requiring urgent action to develop more effective antibiotics. Utilizing the hydrophobic scaffold of xanthone, we identified three components that mimicked the action of an antimicrobial cationic peptide to produce membrane-targeting antimicrobials. Compounds 5c and 6, which contain a hydrophobic xanthone core, lipophilic chains and cationic amino acids, displayed very promising antimicrobial activity against multidrug-resistant Gram-positive bacteria, including MRSA and VRE, rapid time-kill, avoidance of antibiotic resistance and low toxicity. The bacterial membrane selectivity of these molecules was comparable to that of several membrane-targeting antibiotics in clinical trials. 5c and 6 were effective in a mouse model of corneal infection by S. aureus and MRSA. Evidence is presented indicating that 5c and 6 target the negatively charged bacterial membrane via a combination of electrostatic and hydrophobic interactions. These results suggest that 5c and 6 have significant promise for combating life-threatening infections.
This work described how to tune the amphiphilic conformation of α-mangostin, a natural compound with a hydrophobic xanthone scaffold to improve the antimicrobial activity and selectivity for Gram-positive bacteria. A series of xanthone derivatives were obtained by cationic modification of free hydroxyl groups of α-mangostin at C3 and C6 positions with amines groups of different pKa values. Modified structures using high pKa moieties such as AM-0016 (3b) showed potent antimicrobial properties against Gram-positive bacteria. 3b also killed bacteria rapidly, did not induce drug resistance and was non-toxic when applied topically. Biophysical studies and molecular dynamic simulations revealed that 3b targeted the bacterial inner membrane, forming an amphiphilic conformation at the hydrophobic-water interface. In contrast, moieties conjugated to the xanthone scaffold with low pKa values diminished the antimicrobial activities. This strategy provides a new insight to improve "hits" in the development of new membrane-active antibiotics for drug-resistant pathogens.
1H-Pyrazole-1-carboxamidine hydrochloride (1) can be smoothly prepared in excellent yield from readily available starting materials and has stability, reactivity, and solubility properties desirable in a versatile reagent for the efficient and chemically specific guanylation of sterically unhindered primary and secondary aliphatic amines under mild conditions. Unless aromatic amines contain an activating substituent (e.g., 4-methoxyaniline), guanylation by 1 at room temperature is not possible. Other potentially nucleophilic functional groups (hydroxyl, carboxyl, thiol, imidazole) do not observably react with 1 under the mild conditions used for efficient guanylation of simple amines. The feasibility of the use of 1 for the practical synthesis of small peptides by an "ornithine → arginine" strategy has been demonstrated. Additional work is required on more complex peptide targets before the use of 1 for the synthesis of arginine-containing peptides can become generalized and commonplace, although preliminary results suggest the possibility of such an application. Applications of 1 to guanylation of proteins, drugs, drug candidates, natural products, aminopolymers, and in general organic synthesis strategies may also be important.
Persistent infections are frequently caused by dormant and biofilm-associated bacteria, which often display characteristically slow growth. Antibiotics that require rapid cell growth may be ineffective against these organisms and thus fail to prevent reoccurring infections. In contrast to growth-based antimicrobial agents, membrane-targeting drugs effectively kill slow-growing bacteria. Herein we introduce 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane-1,3-diol (DCAP), a potent broad-spectrum antibiotic that reduces the transmembrane potential of Gram-positive and Gram-negative bacteria and causes mislocalization of essential membrane-associated proteins, including MinD and FtsA. Importantly, DCAP kills nutrient-deprived microbes and sterilizes bacterial biofilms. DCAP is lethal against bacterial cells, has no effect on red blood cell membranes, and only decreases the viability of mammalian cells after ≥6 h. We conclude that membrane-active compounds are a promising solution for treating persistent infections. DCAP expands the limited number of compounds in this class of therapeutic small molecules and provides new opportunities for the development of potent broad-spectrum antimicrobial agents.
Antimicrobial peptides (AMPs) take part in the immune system by mounting a first line of defense against pathogens. Recurrent structural and functional aspects are observed among peptides from different sources, particularly the net cationicity and amphipathicity. However, the membrane seems to be the key determinant of their action, either as the main target of the peptide action or by forming a barrier that must be crossed by peptides to target core metabolic pathways. More importantly, the specificity exhibited by antimicrobial peptides relies on the different lipid composition between pathogen and host cells, likely contributing to their spectrum of activity. Several mechanisms of action have been reported, which may involve membrane permeabilization through the formation of pores, membrane thinning or micellization in a detergent-like way. AMPs may also target intracellular components, such as DNA, enzymes and even organelles. More recently, these peptides have been shown to produce membrane perturbation by formation of specific lipid-peptide domains, lateral phase segregation of zwitterionic from anionic phospholipids and even the formation of non-lamellar lipid phases. To countermeasure their activity, some pathogens were successful in developing effective mechanisms of resistance to decrease their susceptibility to AMPs. The functional and integral knowledge of such interactions and the clarification of the complex interplay between molecular determinants of peptides, the pathogen versus host cells dichotomy and the specific microenvironment in which all these elements convene will contribute to an understanding of some elusive aspects of their action and to rationally design novel therapeutic agents to overcome the current antibiotic resistance issue.
Novel compounds termed lipophosphonoxins were prepared using a simple and efficient synthetic approach. The general structure of lipophosphonoxins consists of four modules: (i) a nucleoside module, (ii) an iminosugar module, (iii) a hydrophobic module (lipophilic alkyl chain), and (iv) a phosphonate linker module that holds together modules i-iii. Lipophosphonoxins displayed significant antibacterial properties against a panel of Gram-positive species, including multiresistant strains. The minimum inhibitory concentration (MIC) values of the best inhibitors were in the 1-12 μg/mL range, while their cytotoxic concentrations against human cell lines were significantly above this range. The modular nature of this artificial scaffold offers a large number of possibilities for further modifications/exploitation of these compounds.
Since their introduction in therapy, antibiotics have played an essential role in human society, saving millions of lives, allowing safe surgery, organ transplants, cancer therapy. Antibiotics have also helped to elucidate several biological mechanisms and boosted the birth and growth of pharmaceutical companies, generating profits and royalties. The golden era of antibiotics and the scientific and economical drive of big pharma towards these molecules is long gone, but the need for effective antibiotics is increased as their pipelines dwindle and multi-resistant pathogenic strains spread. Here we outline some strategies that could help meet this emergency and list promising new targets.
Bacteria are known to communicate primarily via secreted extracellular factors. Here we identify a previously uncharacterized type of bacterial communication mediated by nanotubes that bridge neighboring cells. Using Bacillus subtilis as a model organism, we visualized transfer of cytoplasmic fluorescent molecules between adjacent cells. Additionally, by coculturing strains harboring different antibiotic resistance genes, we demonstrated that molecular exchange enables cells to transiently acquire nonhereditary resistance. Furthermore, nonconjugative plasmids could be transferred from one cell to another, thereby conferring hereditary features to recipient cells. Electron microscopy revealed the existence of variously sized tubular extensions bridging neighboring cells, serving as a route for exchange of intracellular molecules. These nanotubes also formed in an interspecies manner, between B. subtilis and Staphylococcus aureus, and even between B. subtilis and the evolutionary distant bacterium Escherichia coli. We propose that nanotubes represent a major form of bacterial communication in nature, providing a network for exchange of cellular molecules within and between species.
Serial passage of Pseudomonas aeruginosa ATCC 27853 or Escherichia coli ATCC 25922 on agar with subinhibitory concentrations of norfloxacin rapidly produced isolates with minimal inhibitory concentrations (MICs) of norfloxacin up to 512-fold higher than that for the original strain. Although MICs of seven unrelated antibiotics were unchanged, increasing MICs occurred in parallel with norfloxacin, cinoxacin, and nalidixic acid regardless of which of these three organic acids was used to select for increased resistance. P. aeruginosa with a norfloxacin MIC of greater than 256 micrograms/ml could be selected; however, E. coli with MICs greater than the clinically achievable level of 16 micrograms/ml could not be produced.
The antibacterial activities of cationic steroid antibiotics and cationic peptide antibiotics have been compared. Depolarization of bacterial membranes, activation of bacterial stress-related gene promoters, and changes in bacterial morphologies caused by these antibiotics suggest that cationic steroid and peptide antibiotics share mechanistic aspects. Modified cationic steroid antibiotics display improved selectivity for prokaryotic cells over eukaryotic cells presumably due to increased charge recognition.
Cracking the case: Aryl amide oligomers with amphiphilic secondary structure were designed that attack bacteria by lysing their membranes. A variety of groups were appended to the lead compound to adjust its overall charge, hydrophobicity, and hydrophobic moment. An Arg-containing oligomer (see figure) was found to have good antimicrobial activity and low toxicity towards human erythrocytes.
Cationic steroid antibiotics have been developed that display broad-spectrum antibacterial activity. These compounds are comprised of steroids appended with amine groups arranged to yield facially amphiphilic morphology. Examples of these antibiotics are highly bactericidal, while related compounds effectively permeabilize the outer membranes of Gram-negative bacteria sensitizing these organisms to hydrophobic antibiotics. Cationic steroid antibiotics exhibit various levels of eukaryote vs. prokaryote cell selectivity, and cell selectivity can be increased via charge recognition of prokaryotic cells. Studies of the mechanism of action of these antibiotics suggest that they share mechanistic aspects with cationic peptide antibiotics.
Melittin is the principal toxic component in the venom of the European honey bee Apis mellifera and is a cationic, hemolytic peptide. It is a small linear peptide composed of 26 amino acid residues in which the amino-terminal region is predominantly hydrophobic whereas the carboxy-terminal region is hydrophilic due to the presence of a stretch of positively charged amino acids. This amphiphilic property of melittin has resulted in melittin being used as a suitable model peptide for monitoring lipid-protein interactions in membranes. In this review, the solution and membrane properties of melittin are highlighted, with an emphasis on melittin-membrane interaction using biophysical approaches. The recent applications of melittin in various cellular processes are discussed.
Ceragenins are cationic bile salt derivatives having antimicrobial activity. The interactions of several ceragenins with phospholipid bilayers were tested in different systems. The ceragenins are capable of forming specific associations with several phospholipid species that may be involved with their antimicrobial action. Their antimicrobial activity is lower in bacteria that have a high content of phosphatidylethanolamine. Gram negative bacteria with a high content of phosphatidylethanolamine exhibit sensitivity to different ceragenins that corresponds to the extent of interaction of these compounds with phospholipids, including the ability of different ceragenins to induce leakage of aqueous contents from phosphatidylethanolamine-rich liposomes. A second class of bacteria having cell membranes composed largely of anionic lipids and having a low content of phosphatidylethanolamine are very sensitive to the action of the ceragenins but they exhibit similar minimal inhibitory concentrations with most of the ceragenins and for different strains of bacteria. Although Gram negative bacteria generally have a high content of phosphatidylethanolamine, there are a few exceptions. In addition, a mutant strain of Escherichia coli has been made that is essentially devoid of phophatidylethanolamine, although 80% of the lipid of the wild-type strain is phosphatidylethanolamine. Furthermore, certain Gram positive bacteria are also exceptions in that they can have a high content of phosphatidylethanolamine. We find that the antimicrobial action of the ceragenins correlates better with the content of phosphatidylethanolamine in the bacterial membrane than whether or not the bacteria has an outer membrane. Thus, the bacterial lipid composition can be an important factor in determining the sensitivity of bacteria to antimicrobial agents.
C 32 H 63 O 8 N 5 P (M+H) + vypoč teno 676
- Hr-Esi
HR-ESI C 32 H 63 O 8 N 5 P (M+H) + vypoč teno 676.44088, nalezeno
676.44092.
Hexadecyl Uridin-5′-yl 2-N-bis(3-aminopropyl)-2-aminoethyl-
2854 (s), 2560 (m, vbr), 2040 (vw, vbr
- Max Ir Ν
IR ν max (KBr) 3427 (s, br), 3000 (s, vbr), 2956 (s), 2924 (vs), 2854
(s), 2560 (m, vbr), 2040 (vw, vbr), 1691 (s), 1466 (m), 1407 (w),
1387 (w), 1267 (m), 1235 (m, br, sh), 1073 (m, sh), 1051 (m, sh),
1018 (m), 1003 (m, sh), 824 (w), 766 (w, sh), 721 (vw).
3018 (s, vbr, sh), 2924 (vs), 2854 (vs), 2650 (m, vbr, sh), 2560 (m, vbr), 2032 (vw, vbr), 1946 (vw, vbr), 1691 (s, br
P{ 1 H} NMR (202.3 MHz, CD 3 OD): 33.66.
ν max (KBr) 3423 (s, vbr), 3018 (s, vbr, sh), 2924 (vs), 2854 (vs),
2650 (m, vbr, sh), 2560 (m, vbr), 2032 (vw, vbr), 1946 (vw, vbr),
1691 (s, br), 1626 (m), 1466 (s), 1406 (m), 1387 (m), 1266 (m),
1237 (m, br, sh), 1074 (m, sh), 1052 (m, sh), 1021 (s, br), 1000 (m,
br, sh), 822 (w), 767 (w), 722 (w).
Journal of Medicinal Chemistry
Article
DOI: 10.1021/acs.jmedchem.7b00355
J. Med. Chem. 2017, 60, 6098−6118
1261 (s, br), 1224 (m), 1075 (s, sh), 1053 (s), 1036 (s, sh), 1019 (s, sh
- Max Ir Ν
IR ν max (CHCl 3 ) 3415 (s, vbr), 3051 (s, br), 2924 (vs), 2854 (vs),
2755 (m, vbr, sh), 2455 (w, vbr), 2030 (vw, vbr), 1970 (vw, vbr), 1693
(vs, br), 1464 (s), 1405 (m, sh), 1385 (m), 1261 (s, br), 1224 (m),
1075 (s, sh), 1053 (s), 1036 (s, sh), 1019 (s, sh), 996 (s), 822 (m),
766 (w), 721 (w).
2960 (s), 2923 (vs), 2853 (vs), 2698 (s, br), 2602 (s, br
- Max Ir Ν
IR ν max (KBr) 3353 (s, vbr), 3037 (s, vbr), 2960 (s), 2923 (vs), 2853
(vs), 2698 (s, br), 2602 (s, br), 2527 (m, br), 2080 (vw, vbr), 1691 (vs,
br), 1465 (m), 1408 (m), 1387 (m), 1267 (s), 1232 (m, br), 1075 (m,
sh), 1055 (s, sh) 1032 (s, br) 996 (s), 764 (w), 721 (w).
C 35 H 63 O 9 N 5 P (M+H + ) calcd 630.37575, found 631
- Hr-Esi
HR-ESI C 35 H 63 O 9 N 5 P (M+H + ) calcd 630.37575, found 631.38299.
Pentadecyl-uridin-5′-yl-((3R,4S)-4-amino-3-hydroxypyrrolidin-1-
2925 (s), 2854 (s), 2710 (m, vbr), 2604 (m), 2502 (m, vbr, sh), 1669 (vs, vbr), 1622 (vs, sh), 1465 (s), 1407 (m), 1379 (s), 1265 (s), 1235 (s, br, sh), 1075 (s, br, sh), 1045 (s), 1016 (s, br), 1002 (s, sh
- Max Ir Ν
IR ν max (KBr) 3320 (s, vbr), 3260 (s, vbr), 3155 (s, vbr), 2925 (s),
2854 (s), 2710 (m, vbr), 2604 (m), 2502 (m, vbr, sh), 1669 (vs, vbr),
1622 (vs, sh), 1465 (s), 1407 (m), 1379 (s), 1265 (s), 1235 (s, br, sh),
1075 (s, br, sh), 1045 (s), 1016 (s, br), 1002 (s, sh), 822 (m), 720 (w),
580 (m, vbr), 490 (m, br, sh).
2956 (s), 2923 (s), 2853 (s), 2609 (m, vbr), 1694 (vs, sh), 1672 (vs, br), 1645 (vs, sh
- Max Ir Ν
IR ν max (KBr) 3337 (s, vbr), 3167 (s, br), 2956 (s), 2923 (s), 2853
(s), 2609 (m, vbr), 1694 (vs, sh), 1672 (vs, br), 1645 (vs, sh), 1465
(s), 1408 (m), 1387 (m), 1264 (s), 1229 (s), 1074 (s, sh), 1054 (s),
1023 (s), 1020 (s), 995 (s), 762 (vw), 720 (w).
C 33 H 65 O 8 N 9 P (M+H) + calcd 746.46882, found 746.46916. Tetradecyl 5-(Fur-2-yl)uridin-5′-yl-2-N-bis(3-aminopropyl
- Hr-Esi
HR-ESI C 33 H 65 O 8 N 9 P (M+H) + calcd 746.46882, found 746.46916.
Tetradecyl 5-(Fur-2-yl)uridin-5′-yl-2-N-bis(3-aminopropyl)-2-ami-
Design and synthesis of amphiphilic xanthone-based, membrane-targeting antimicrobials with improved membrane selectivity
- S Tsipshtein
- A Rouvinski
- A Rosenberg
- D Kaganovich
- E Sherman
- O Medalia
- S Ben-Yehuda
- T Zhang
- J K Muraih
- B Maccormick
- J Silverman
- M Palmer
- H Raghuraman
- A Chattopadhyay
- H Zou
- J.-J Koh
- J Li
- S Qiu
- T T Aung
- H Lin
- R Lakshminarayanan
- X Dai
- C Tang
- F H Lim
- L Zhou
- A L Tan
- C Verma
- D T H Tan
- H S O Chan
- P Saraswathi
- D Cao
- S Liu
- R W Beuerman
- C.-H Chuang
- R P Janapatla
- Y.-H Wang
- H.-J Chang
- Y.-C Huang
- T.-Y Lin
- C.-H Chiu
Tsipshtein, S.; Rouvinski, A.; Rosenberg, A.; Kaganovich, D.; Sherman,
E.; Medalia, O.; Ben-Yehuda, S. Architecture and characteristics of
bacterial nanotubes. Dev. Cell 2016, 36, 453−461.
(22) Zhang, T.; Muraih, J. K.; MacCormick, B.; Silverman, J.; Palmer,
M. Daptomycin forms cation-and size-selective pores in model
membranes. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 2425−
2430.
(23) Raghuraman, H.; Chattopadhyay, A. Melittin: a membraneactive peptide with diverse functions. Biosci. Rep. 2007, 27, 189−223.
(24) Zou, H.; Koh, J.-J.; Li, J.; Qiu, S.; Aung, T. T.; Lin, H.;
Lakshminarayanan, R.; Dai, X.; Tang, C.; Lim, F. H.; Zhou, L.; Tan, A.
L.; Verma, C.; Tan, D. T. H.; Chan, H. S. O.; Saraswathi, P.; Cao, D.;
Liu, S.; Beuerman, R. W. Design and synthesis of amphiphilic
xanthone-based, membrane-targeting antimicrobials with improved
membrane selectivity. J. Med. Chem. 2013, 56, 2359−2373.
(25) McConnell, E. L.; Basit, A. W.; Murdan, S. Measurements of rat
and mouse gastrointestinal pH, fluid and lymphoid tissue, and
implications for in-vivo experiments. J. Pharm. Pharmacol. 2008, 60,
63−70.
(26) http://www.fgu.cas.cz/en/departments/biological-controls, January 17, 2017.
(27) Chuang, C.-H.; Janapatla, R. P.; Wang, Y.-H.; Chang, H.-J.;
Huang, Y.-C.; Lin, T.-Y.; Chiu, C.-H. Pseudomonas aeruginosaassociated diarrheal diseases in children. Pediatr. Infect. Dis. J. [Online
early access]. DOI: 10.1097/INF.0000000000001567 Published
online: Jan 31, 2017. http://journals.lww.com/pidj/Abstract/
publishahead/Pseudomonas_aeruginosa_Associated_Diarrheal.
97084.aspx (accessed Feb. 12, 2017).
DMSO-d 6 ): 0.90 (m, 6H, CH 3 (CH 2 ) 13 CH 2 O)
- H Nmr
H NMR (500.0 MHz, DMSO-d 6 ): 0.90 (m, 6H,
CH 3 (CH 2 ) 13 CH 2 O); 1.24−1.43 (m, 48H, CH 3 (CH 2 ) 12 CH 2 CH 2 O);
7 MHz, DMSO-d 6 ): 14.46 (CH 3 (CH 2 ) 13 CH 2 O)
- C Nmr
C NMR (125.7 MHz, DMSO-d 6 ): 14.46 (CH 3 (CH 2 ) 13 CH 2 O);
41 (PCH 2 CH 2 N-A); 51.64 (NCH 2 CH 2 CH 2 NH-A,B); 67.36 (d, J C,P = 6.6, CH 2 −5′-A); 67.45 (d
- B Nh-A
39.66 (NCH 2 CH 2 CH 2 NH-A,B); 48.38 (PCH 2 CH 2 N−B); 48.41
(PCH 2 CH 2 N-A); 51.64 (NCH 2 CH 2 CH 2 NH-A,B); 67.36 (d, J C,P =
6.6, CH 2 −5′-A); 67.45 (d, J C,P = 6.4, CH 2 −5′-B); 68.33 (d, J C,P = 6.9,
C H 3 ( C H 2 d, J C, P = 6.8,
CH 3 (CH 2 ) 10 CH 2 CH 2 O-A); 70.82 (CH-3′-B); 70.87 (CH-3′-A);
38 (PCH 2 CH 2 N-A,B); 51.56 (NCH 2 CH 2 CH 2 NH-A,B); 67.31 (d, J C,P = 6.5, CH 2 −5′-B); 67.37 ( d , J C , P = 6.5, CH 2 − 5 ′ -A ) ; d , J C , P = 6.8, CH 3 (CH 2 ) 11 CH 2 CH 2 O−B)
- B Nh-A
39.64 (NCH 2 CH 2 CH 2 NH-A,B); 48.38 (PCH 2 CH 2 N-A,B); 51.56
(NCH 2 CH 2 CH 2 NH-A,B); 67.31 (d, J C,P = 6.5, CH 2 −5′-B); 67.37
( d, J C, P = 6.5, CH 2 − 5 ′ -A ) ; d, J C, P = 6.8,
CH 3 (CH 2 ) 11 CH 2 CH 2 O−B); 70.78 (CH-3′-A); 70.84 (CH-3′-B);
Measurements of rat and mouse gastrointestinal pH, fluid and lymphoid tissue, and implications for in-vivo experiments
- E L Mcconnell
- A W Basit
- S Murdan
- C.-H Chuang
- R P Janapatla
- Y.-H Wang
- H.-J Chang
- Y.-C Huang
- T.-Y Lin
- C.-H Chiu
McConnell, E. L.; Basit, A. W.; Murdan, S. Measurements of rat
and mouse gastrointestinal pH, fluid and lymphoid tissue, and
implications for in-vivo experiments. J. Pharm. Pharmacol. 2008, 60,
63−70.
(26) http://www.fgu.cas.cz/en/departments/biological-controls, January 17, 2017.
(27) Chuang, C.-H.; Janapatla, R. P.; Wang, Y.-H.; Chang, H.-J.;
Huang, Y.-C.; Lin, T.-Y.; Chiu, C.-H. Pseudomonas aeruginosaassociated diarrheal diseases in children. Pediatr. Infect. Dis. J. [Online
early access]. DOI: 10.1097/INF.0000000000001567 Published
online: Jan 31, 2017. http://journals.lww.com/pidj/Abstract/
publishahead/Pseudomonas_aeruginosa_Associated_Diarrheal.
97084.aspx (accessed Feb. 12, 2017).
Lipophosphonoxins, method of their preparation and use
- D Rejman
- R Pohl
- P Bartunek
- A J Ribeiro Pombinho
- L Krasny
- T Latal
Rejman, D.; Pohl, R.; Bartunek, P.; Ribeiro Pombinho, A. J.;
Krasny, L.; Latal, T. Lipophosphonoxins, method of their preparation
and use. EP 2 527 351 B1, 11.12.2013, 2013.