Thet Tun Aung

Amino acid modified xanthone derivatives: novel, highly promising membrane-active antimicrobials for multidrug-resistant Gram-positive bacterial infections.

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.

Synthetic Multivalent Antifungal Peptides Effective against Fungi

Taking advantage of the cluster effect observed in multivalent peptides, this work describes antifungal activity and possible mechanism of action of tetravalent peptide (B4010) which carries 4 copies of the sequence RGRKVVRR through a branched lysine core. B4010 displayed better antifungal properties than natamycin and amphotericin B. The peptide retained significant activity in the presence of monovalent/divalent cations, trypsin and serum and tear fluid. Moreover, B4010 is non-haemolytic and non-toxic to mice by intraperitoneal (200 mg/kg) or intravenous (100 mg/kg) routes. S. cerevisiae mutant strains with altered membrane sterol structures and composition showed hyper senstivity to B4010. The peptide had no affinity for cell wall polysaccharides and caused rapid dissipation of membrane potential and release of vital ions and ATP when treated with C. albicans. We demonstrate that additives which alter the membrane potential or membrane rigidity protect C. albicans from B4010-induced lethality. Calcein release assay and molecular dynamics simulations showed that the peptide preferentially binds to mixed bilayer containing ergosterol over phophotidylcholine-cholesterol bilayers. The studies further suggested that the first arginine is important for mediating peptide-bilayer interactions. Replacing the first arginine led to a 2–4 fold decrease in antifungal activities and reduced membrane disruption properties. The combined in silico and in vitro approach should facilitate rational design of new tetravalent antifungal peptides.

N-Lipidated Peptide Dimers: Effective Antibacterial Agents against Gram-Negative Pathogens through Lipopolysaccharide Permeabilization

Treating infections caused by multidrug-resistant Gram-negative pathogens is challenging, and there is concern regarding the toxicity of the most effective antimicrobials for Gram-negative pathogens. We hypothesized that conjugating a fatty acid moiety onto a peptide dimer could maximize the interaction with lipopolysaccharide (LPS) and facilitate the permeabilization of the LPS barrier, thereby improving potency against Gram-negative pathogens. We systematically designed a series of N-lipidated peptide dimers that are active against Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae (CRE). The optimized lipid length was 6-10 carbons. At these lipid lengths, the N-lipidated peptide dimers exhibited strong LPS permeabilization. Compound 23 exhibited synergy with select antibiotics in most of the combinations tested. 23 and 32 also displayed rapid bactericidal activity. Importantly, 23 and 32 were nonhemolytic at 10 mg/mL, with no cellular or in vivo toxicity. These characteristics suggest that these compounds can overcome the limitations of current Gram-negative-targeted antimicrobials such as polymyxin B.

Biofilms of pathogenic nontuberculous mycobacteria targeted by new therapeutic approaches

ABSTRACT Microbial infections of the cornea are potentially devastating and can result in permanent visual loss or require vision-rescuing surgery. In recent years, there has been an increasing number of reports on nontuberculous mycobacterial infections of the cornea. Challenges to the management of nontuberculous mycobacterial keratitis include delayed laboratory detection, low index of clinical suspicion, poor drug penetration, slow response to therapy, and prolonged use of antibiotic combinations. The ability of nontuberculous mycobacteria to evade the host immune response and the ability to adhere and to form biofilms on biological and synthetic substrates contribute to the issue. Therefore, there is an urgent need for new antimicrobial compounds that can overcome these problems. In this study, we evaluated the biofilm architectures for Mycobacterium chelonae and Mycobacterium fortuitum in dynamic flow cell chamber and 8-well chamber slide models. Our results showed that mycobacterial biofilms were quite resistant to conventional antibiotics. However, DNase treatment could be used to overcome biofilm resistance. Moreover, we successfully evaluated a new antimicrobial compound (AM-228) that was effective not only for planktonic mycobacterial cells but also for biofilm treatment and was compared favorably with the most successful “fourth-generation” fluoroquinolone, gatifloxacin. Finally, a new treatment strategy emerged: a combination of DNase with an antibiotic was more effective than an antibiotic alone.

Symmetrically Substituted Xanthone Amphiphiles Combat Gram-Positive Bacterial Resistance with Enhanced Membrane Selectivity

This is the first report of the design of a new series of symmetric xanthone derivatives that mimic antimicrobial peptides using a total synthesis approach. This novel design is advantageous because of its low cost, synthetic simplicity and versatility, and easy tuning of amphiphilicity by controlling the incorporated cationic and hydrophobic moieties. Two water-soluble optimized compounds, 6 and 18, showed potent activities against Gram-positive bacteria, including MRSA and VRE (MICs = 0.78-6.25 μg/mL) with a rapid bactericidal effect, low toxicity, and no emergence of drug resistance. Both compounds demonstrated enhanced membrane selectivity that was higher than those of most membrane-active antimicrobials in clinical trials or previous reports. The compounds appear to kill bacteria by disrupting their membranes. Significantly, 6 was effective in vivo using a mouse model of corneal infection. These results provide compelling evidence that these compounds have therapeutic potential as novel antimicrobials for multidrug-resistant Gram-positive infections.

Semisynthetic Flavone-Derived Antimicrobials with Therapeutic Potential against Methicillin-Resistant Staphylococcus aureus (MRSA)

A new series of semisynthetic flavone-based small molecules mimicking antimicrobial peptides has been designed from natural icaritin to combat drug-resistant Gram-positive bacterial infections. Compound 6 containing two arginine residues exhibited excellent antibacterial activity against Gram-positive bacteria, including MRSA, and very low toxicity to mammalian cells, resulting in a high selectivity of more than 511, comparable to that of several membrane-active antibiotics in clinical trials. Our data show for the first time that icaritin derivatives effectively kill bacteria. Meanwhile, this is the first study deploying a biomimicking strategy to design potent flavone-based membrane targeting antimicrobials. 6 showed rapid bactericidal activity by disrupting the bacterial membrane and can circumvent the development of bacterial resistance. Importantly, 6 was highly efficacious in a mouse model of corneal infection caused by MRSA and Staphylococcus aureus.

Branched Peptide, B2088, Disrupts the Supramolecular Organization of Lipopolysaccharides and Sensitizes the Gram-negative Bacteria

Dissecting the complexities of branched peptide-lipopolysaccharides (LPS) interactions provide rationale for the development of non-cytotoxic antibiotic adjuvants. Using various biophysical methods, we show that the branched peptide, B2088, binds to lipid A and disrupts the supramolecular organization of LPS. The disruption of outer membrane in an intact bacterium was demonstrated by fluorescence spectroscopy and checkerboard assays, the latter confirming strong to moderate synergism between B2088 and various classes of antibiotics. The potency of synergistic combinations of B2088 and antibiotics was further established by time-kill kinetics, mammalian cell culture infections model and in vivo model of bacterial keratitis. Importantly, B2088 did not show any cytotoxicity to corneal epithelial cells for at least 96 h continuous exposure or hemolytic activity even at 20 mg/ml. Peptide congeners containing norvaline, phenylalanine and tyrosine (instead of valine in B2088) displayed better synergism compared to other substitutions. We propose that high affinity and subsequent disruption of the supramolecular assembly of LPS by the branched peptides are vital for the development of non-cytotoxic antibiotic adjuvants that can enhance the accessibility of conventional antibiotics to the intracellular targets, decrease the antibiotic consumption and holds promise in averting antibiotic resistance.

Moesin expression in fibrosis in the mouse cornea after sterile mechanical trauma or infection.

Purpose: The aim of this study was to compare the expression patterns of 3 important biochemical characteristics of fibrosis–moesin, transforming growth factor (TGF)-&bgr;1, and &agr;-smooth muscle actin (SMA) in the mouse cornea with fibrosis induced by common etiologies—sterile mechanical injury and infection. Methods: Corneas of 8-week-old C57BL6 mice were either wounded after an anterior keratectomy or were infected by Pseudomonas aeruginosa, and the animals were killed on days 2 and 7, and on weeks 2 and 4 after the procedure. Western blot and immunofluorescence were used to analyze the expression of moesin and phospho-moesin, and the presence of myofibroblasts identified by the expression of &agr;-SMA in the corneal stroma. The expression of TGF-&bgr;1 was analyzed by immunofluorescence. Results: By immunofluorescent analysis, TGF-&bgr;1, &agr;-SMA, and phospho-moesin were not detected in the normal corneal stroma. However, after either treatment, TGF-&bgr;1 expression increased, along with phospho-moesin in the wounded corneal stroma until day 7, and decreased after week 2. No expression of TGF-&bgr;1 and phospho-moesin was found at postoperative week 4. Moesin expression increased until week 2. Myofibroblasts positive for &agr;-SMA were detected on day 2 until week 4 and peaked at week 2. Western blot analysis confirmed the immunofluorescent data for moesin, phospho-moesin, and &agr;-SMA. Conclusions: The similar expression pattern of moesin, phospho-moesin, TGF-&bgr;1, and &agr;-SMA in the mouse cornea with fibrosis caused by sterile mechanical injury or infection indicated a role for moesin signaling in corneal fibrosis. Interference with the action of moesin may be a potential approach for intervention strategies to avert fibrosis after infection or mechanical injury.

Antimicrobial Activity and Cell Selectivity of Synthetic and Biosynthetic Cationic Polymers

ABSTRACT The mammalian and microbial cell selectivity of synthetic and biosynthetic cationic polymers has been investigated. Among the polymers with peptide backbones, polymers containing amino side chains display greater antimicrobial activity than those with guanidine side chains, whereas ethylenimines display superior activity over allylamines. The biosynthetic polymer ε-polylysine (εPL) is noncytotoxic to primary human dermal fibroblasts at concentrations of up to 2,000 μg/ml, suggesting that the presence of an isopeptide backbone has greater cell selectivity than the presence of α-peptide backbones. Both εPL and linear polyethylenimine (LPEI) exhibit bactericidal properties by depolarizing the cytoplasmic membrane and disrupt preformed biofilms. εPL displays broad-spectrum antimicrobial properties against antibiotic-resistant Gram-negative and Gram-positive strains and fungi. εPL elicits rapid bactericidal activity against both Gram-negative and Gram-positive bacteria, and its biocompatibility index is superior to those of cationic antiseptic agents and LPEI. εPL does not interfere with the wound closure of injured rabbit corneas. In a rabbit model of bacterial keratitis, the topical application of εPL (0.3%, wt/vol) decreases the bacterial burden and severity of infections caused by Pseudomonas aeruginosa and Staphylococcus aureus strains. In vivo imaging studies confirm that εPL-treated corneas appeared transparent and nonedematous compared to untreated infected corneas. Taken together, our results highlight the potential of εPL in resolving topical microbial infections.

Antimicrobial activity profiles of Amphiphilic Xanthone derivatives are a function of their molecular Oligomerization

Currently, membrane-targeting small antimicrobial peptidomimetics (SAP) are important in antibiotic development because bacteria appear to develop resistance to these surface-active compounds less readily. However, the molecular membrane-targeting action of SAPs has received little attention. In this study, we investigated the effect of oligomerization of amphiphilic xanthone, a model SAP, on its antimicrobial properties against both Gram-positive and Gram-negative bacteria. First, oligomer formation by an amphiphilic xanthone, compound 2 (also coded as AM052), was investigated via solution-state nuclear magnetic resonance (NMR) spectroscopy. Then, the effects of oligomerization on membrane disruption were further studied via biophysical approaches. The results showed that the antimicrobial activities of SAPs develop in several stages: oligomer formation in aqueous solution, initial binding of oligomers to the membrane-water interface followed by insertion into the membrane bilayer, aggregation of antimicrobial oligomers in the membrane, and induced membrane leakage. Ultimately, the presence of the oligomers in the bacterial membrane leads to decreased membrane fluidity and bacterial cell death. Interestingly, the early formation of large oligomers leads to stronger membrane disruption and more rapid bacterial killing. However, reduced antimicrobial activities against Gram-negative bacteria were observed for compounds that formed larger oligomers because the LPS layer acts as a barrier to large complexes. Taken together, our results suggest that oligomerization of SAPs has a strong impact on their antimicrobial properties.