Antibacterial properties of dermaseptin S4 derivatives under extreme incubation conditions

Antibacterial properties of the frog-derived peptide dermaseptin S4 and a series of synthetic derivatives against the food pathogen Escherichia coli O157:H7 were investigated under extreme incubation conditions. The 28-mer analog K4K20S4 (P28) displayed an MIC of 8 microM and rapid bactericidal kinetics under standard culture conditions. Potent bactericidal properties were maintained at high salt concentrations, under acidic or basic conditions, and at extreme temperatures. The N-terminal 14-mer sequence (P14) displayed higher potency (MIC, 4 microM) but only within a narrow range of incubation conditions, pointing to the importance of the C-terminal domain of P28. The potency range was reextended upon conjugation of aminododecanoic acid to P14. The resulting lipopeptide was even more potent (MIC, 2 microM) and affected bacterial viability under most of the conditions tested, including in commercial apple juice. The mechanistic implications of peptides' hydrophobicity, charge, structure, and binding to an idealized membrane were probed and are discussed here. Collectively, the data indicate interest in simple peptide-based compounds for design of antimicrobials that affect pathogens under a variable range of incubation conditions.     

Antimalarial activities of dermaseptin S4 derivatives

The hemolytic antimicrobial peptide dermaseptin S4 was recently shown to exert antimalarial activity. In this study, we attempted to understand the underlying mechanism(s) and identify derivatives with improved antimalarial activity. A number of dermaseptin S4 derivatives inhibited parasite growth with a 50% inhibitory concentration (IC(50)) in the micromolar range. Among these, the substituted S4 analog K(4)K(20)-S4 was the most potent (IC(50) = 0.2 microM), while its shorter version, K(4)-S4(1-13)a, retained a considerable potency (IC(50) = 6 microM). Both K(4)K(20)-S4 and K(4)-S4(1-13)a inhibited growth of the parasites more at the trophozoite stage than at the ring stage. Significant growth inhibition was observed after as little as 1 min of exposure to peptides and proceeded with nearly linear kinetics. The peptides selectively lysed infected red blood cells (RBC) while having a weaker effect on noninfected RBC. Thus, K(4)K(20)-S4 lysed trophozoites at concentrations similar to those that inhibited their proliferation, but trophozoites were >30-fold more susceptible than normal RBC to the lytic effect of K(4)K(20)-S4, the most hemolytic dermaseptin. The same trend was observed with K(4)-S4(1-13)a. The D isomers of K(4)K(20)-S4 or K(4)-S4(1-13)a were as active as the L counterparts, indicating that antimalarial activity of these peptides, like their membrane-lytic activity, is not mediated by specific interactions with a chiral center. Moreover, dissipation of transmembrane potential experiments with infected cells indicated that the peptides induce damage in the parasite's plasma membrane. Fluorescence confocal microscopy analysis of treated infected cells also indicated that the peptide is able to find its way through the complex series of membranes and interact directly with the intracellular parasite. Overall, the data showed that dermaseptins exert antimalarial activity by lysis of infected cells. Dermaseptin derivatives are also able to disrupt the parasite plasma membrane without harming that of the host RBC.     

Acyl-substituted dermaseptin S4 derivatives with improved bactericidal properties, including on oral microflora

The 15-mer dermaseptin S4 derivative S4(1-15) was recently shown to exhibit potent activity against oral pathogens associated with caries and periodontitis. Here, we investigated possible modes for improving the peptide's properties through systematic replacement of an N-terminal amino acid(s) with various fatty acids that modulate the peptide's hydrophobicity and/or charge. Deletion of 1 to 3 residues led to progressive loss of potency as assessed by MIC experiments performed on four test bacteria. Replacing the deleted amino acids with fatty acids most often resulted in potency recovery or improvement, as evidenced by lower MICs and faster bactericidal kinetics in culture media. Best results were obtained after replacement of the N-terminal dipeptide alanine-leucine with heptanoic (C7) or aminododecanoic (NC12) acid. Circular dichroism analysis correlated antibacterial properties to the peptide's secondary structure. MIC experiments and confocal laser scanning microscopy results indicated that C7-S4(3-15) and NC12-S4(3-15) were bactericidal to various oral pathogens, including those which are immobilized in a biofilm. C7-S4(3-15) performed similarly to or better than (depending on growth medium) IB-367, a peptide assessed in clinical trials for treatment of oral mucositis, reducing CFU counts by >3 log units within 2 min of incubation. Collectively, the data indicate that substitution of fatty acids for amino acids may be a useful strategy in revealing improved derivatives of known antimicrobial peptides and suggest the suitability of such compounds for controlling pathogens associated with oral diseases.     

In vitro antiplasmodium effects of dermaseptin S4 derivatives

The 13-residue dermaseptin S4 derivative K(4)S4(1-13)a (P) was previously shown to kill intraerythrocytic malaria parasites through the lysis of the host cells. In this study, we have sought peptides that will kill the parasite without lysing the erythrocyte. To produce such peptides, 26 compounds of variable structure and size were attached to the N terminus of P and screened for antiplasmodium and hemolytic activities in cultures of Plasmodium falciparum. Results from this screen indicated that increased hydrophobicity results in amplified antiplasmodium effect, irrespective of the linearity or bulkiness of the additive. However, increased hydrophobicity also was generally associated with increased hemolysis, with the exception of two derivatives: propionyl-P (C3-P) and isobutyryl-P (iC4-P). Both acyl-peptides were more effective than P, with 50% growth inhibition at 3.8, 4.3, and 7.7 microM, respectively. The antiparasitic effect was time dependent and totally irreversible, implying a cytotoxic effect. The peptides were also investigated in parallel for their ability to inhibit parasite growth and to induce hemolysis in infected and uninfected erythrocytes. Whereas the dose dependence of growth inhibition and hemolysis of infected cells overlapped when cells were treated with P, the acyl-peptides exerted 50% growth inhibition at concentrations that did not cause hemolysis. Noticeably, the acyl derivatives, but not P, were able to dissipate the parasite plasma membrane potential and cause depletion of intraparasite potassium under nonhemolytic conditions. These results clearly demonstrate that the acyl-peptides can affect parasite viability in a manner that is dissociated from lysis of the host cell. Overall, the data indicate the potential usefulness of this strategy for development of selective peptides as investigative tools and eventually as antimalarial agents.     

Physicochemical properties that enhance discriminative antibacterial activity of short dermaseptin derivatives

Antimicrobial peptides are widely believed to exert their effects by nonspecific mechanisms. We assessed the extent to which physicochemical properties can be exploited to promote discriminative activity by manipulating the N-terminal sequence of the 13-mer dermaseptin derivative K(4)-S4(1-13) (P). Inhibitory activity determined in culture media against 16 strains of bacteria showed that when its hydrophobicity and charge were changed, P became predominantly active against either gram-positive or gram-negative bacteria. Thus, conjugation of various aminoacyl-lysin moieties (e.g., aminohexyl-K-P) led to inactivity against gram-positive bacteria (MIC(50) > 50 microM) but potent activity against gram-negative bacteria (MIC(50), 6.2 microM). Conversely, conjugation of equivalent acyls to the substituted analog M(4)-S4(1-13) (e.g., hexyl-M(4)-P) led to inactivity against gram-negative bacteria (MIC(50) > 50 microM) but potent activity against gram-positive bacteria (MIC(50), 3.1 microM). Surface plasmon resonance experiments, used to investigate peptides' binding properties to lipopolysaccharide-containing idealized phospholipid membranes, suggest that although the acylated derivatives have increased lipophilic properties with parallel antibacterial behavior, hydrophobic derivatives are prevented from reaching the cytoplasmic membranes of gram-negative bacteria. Moreover, unlike modifications that enhanced the activity against gram-positive bacteria, which also enhanced hemolysis, we found that modifications that enhanced activity against gram-negative bacteria generally reduced hemolysis. Thus, compared with the clinically tested peptides MSI-78 and IB-367, the dermaseptin derivative aminohexyl-K-P performed similarly in terms of potency and bactericidal kinetics but was significantly more selective in terms of discrimination between bacteria and human erythrocytes. Overall, the data suggest that similar strategies maybe useful to derive potent and safe compounds from known antimicrobial peptides.     

Antibacterial activity of ofloxacin and other 4-quinolone derivatives: in-vitro and in-vivo comparison

Ofloxacin (DL8280, RU43280) is a newly introduced oxazine quinolone derivative with broad and potent antibacterial activity. Ofloxacin showed excellent in-vitro activity against Enterobacteriaceae while most strains of Pseudomonas aeruginosa were inhibited by less than 2 mg/l. The compound was significantly more potent than norfloxacin against Acinetobacter spp. and Staphylococcus spp. Ofloxacin and norfloxacin behaved similarly with respect to inoculum size, effect of urine and serum, bactericidal properties and frequency of spontaneous resistant mutants. Ofloxacin displayed an in-vivo antibacterial activity up to five times greater than that of pefloxacin and norfloxacin, probably due to the conjunction of favourable pharmacokinetics, excellent bacterial susceptibility and good stability towards metabolic degradation.     

Antibacterial activity evaluation and mode of action study of novel thiazole-quinolinium derivatives

New antimicrobial agents are urgently needed to address the emergence of multi-drug resistant organisms, especially those active compounds with new mechanisms of action. Based on the molecular structures of the FtsZ inhibitors reported, a variety of thiazole-quinolinium derivatives with aliphatic amino and/or styrene substituents were synthesized from benzothiazolidine derivatives. In the present study, to further explore the antibacterial potential of thiazole-quinolinium derivatives, several Gram-positive and Gram-negative bacteria were treated with the newly modified compounds and the biological effects were studied in detail in order to understand the bactericidal action of the compounds. Our findings reveal that some of these derivatives possess good potent bactericidal activity as they can inhibit Gram-positive methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and also some Gram-negative organisms and NDM-1 Escherichia coli. Furthermore, compounds 4a1–4a4 and 4b1–4b4 altered the morphology of bacterial cells and the cells displayed a more-elongated shape compared to the untreated cells. Biochemical assays showed that 4a4 and 4b4 stimulate FtsZ polymerization in bacterial cells, which eventually disrupts its dynamic assembly and Z-ring formation. The inhibition of this crucial step in bacterial cell division could potentially represent their main mechanism of antibacterial activity. Cytotoxicity assay and hemolysis assay suggested that 4a4 and 4b4 possess low cytotoxicity. In summary, these results further highlight the importance of 4a4 and 4b4 that could be developed as potent and effective bacteriostatic agents against multi-drug resistant bacteria.

A new series of thiazole-quinolinium derivatives perturb the polymerization of FtsZ with strong antibacterial activities.     

Antibacterial activity of 2',3'-dideoxyadenosine in vivo and in vitro.

2',3'-Dideoxyadenosine (DDA) was shown not only to possess antibacterial activity in vitro against a variety of Enterobacteriaceae, but also to be effective in vivo, DDA was active in experimental mouse infections by the oral route against 5 Salmonella strains, 2 of 3 Arizona strains, 5 of 7 Citrobacter strains, 3 of 8 Klebsiella strains, 3 of 5 Escherichia strains, 1 of 3 Shigella strains, and 3 of 15 Serratia strains at concentrations generally well below the toxic level. Closely related compounds, with the exception of 2',3'-dideoxyinosine, were found to be inactive in vivo, indicating that a high degree of structural specificity was required for activity. The synthesis of deoxyribonucleic acid was inhibited by DDA in those strains susceptible in vitro to DDA, whereas ribonucleic acid and protein syntheses were not affected. The concentration of DDA which inhibited bacterial deoxyribonucleic acid synthesis by 50% was calculated based on the relative rates of deoxyribonucleic acid synthesis in ;the absence and in the presence of DDA. This value correlated well with the minimal inhibitory concentration determined by the in vitro broth dilution assay but not always with in vivo activity determined by the mouse protection test.     

Antibacterial activities of nitrothiazole derivatives

A new group of chemical agents with pronounced antibacterial activities is presented. Different nitrothiazole derivatives, but not all, exhibit antibacterial activities similar to that of niridazole (AmbilharR), a nitrothiazolyl-imidazolidinone. Certain aerobic bacteria are moderately susceptible to these agents. The efficacy of these agents is almost comparable to that of ampicillin and tetracyline. It is much better than the efficacy of nitrofuran derivatives, the nitroimidazole derivatives being inactive. The mode of action of the nitrothiazole derivatives is bactericidal. Against anaerobic bacteria the nitrothiazole derivatives are unique, since the extremely low MIC's are not approached by any other of the nitro-compounds nor by any other of the common antibiotics tested, such as clindamycin, ampicillin and tetracycline. It is suggested that the nitrogroup of the nitrothiazole ring moiety represents the chemical structure responsible for their excellent antibacterial activities.     

Structure activity relationship of phosphoramidon derivatives for in vivo endothelin-converting-enzyme inhibition

Summary— The structure activity relationship of phosphoramidon analogues was studied for their ability to reduce the hypertensive effect of exogenous proET-1, probably via inhibition of an endothelin converting enzyme activity (ECE). Results concerning in vivo ECE and in vitro thermolysin inhibitions were compared. In contrast to the phosphoryl group of phosphoramidon, which was found to be an absolute requirement, the rhamnose moiety was of very little importance for the inhibition of either enzyme. Furthermore, the tryptophan residue of phosphoramidon appeared to be particularly important for the ECE inhibition, whereas thermolysin inhibition seemed to depend greatly on the leucine residue. It is concluded that in vivo ECE and thermolysin differ in the way they recognise phosphoramidon. The existence of an hydrophobic pocket, specific for the recognition of the tryptophan residue of phosphoramidon, could be proposed for ECE.     

Antibacterial activity and nephrotoxicity of two novel 2″-amino derivatives of arbekacin

The antibacterial activity of 2″-amino-2″-deoxyarbekacin (AmABK) and 2″-amino-5,2″-dideoxy-5-epiaminoarbekacin (Am₂ABK) was comparable to, or slightly less than, that of arbekacin (ABK) against gram-positive and gram-negative bacteria, including 60 stock cultures and 50 clinical isolates ofPseudomonas aeruginosa, but more potent against 31 isolates of MRSA possessing an aminoglycoside-modifying enzyme APH(2″)/AAC(6'). AmABK and Am₂ABK showed in vivo activity which paralleled in vitro MICs, and were less toxic than ABK in acute toxicity in mice and nephrotoxicity in rats. These results indicate that the 2″-amino group introduced to ABK confers high stabilization to the aminoglycoside-modifying enzymes, while reducing acute and renal toxicities.     

Antibacterial properties of gemifloxacin and trovafloxacin in urine ex vivo: phase I study

Urine bactericidal titers (UBTs) against Escherichia coli ATCC 25922 and Staphylococcus saprophyticus ATCC 1970 were determined after the administration of single oral doses of gemifloxacin at 320 mg and trovafloxacin at 200 mg to healthy volunteers. Gemifloxacin presented significantly lower experimental versus mathematically predicted UBTs over 72 h, due to the effect of urine on the susceptibility of the E. coli strain. Experimental UBTs were significantly higher for gemifloxacin than trovafloxacin against both strains over 72 h.     

Electrospun cellulose acetate doped with astaxanthin derivatives from Haematococcus pluvialis for in vivo anti-aging activity

This research aims to study the release, in vivo anti-aging activity against Caenorhabditis elegans and stability of astaxanthins in a crude acetone extract of Haematococcus pluvialis from electrospun cellulose acetate (CA) nanofibers. The content and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging activity of astaxanthins in the crude extract were also determined. The content of astaxanthins was reported in terms of total carotenoid content (TCC) and found to be 10.75 ± 0.16 mg gcE⁻¹. IC₅₀ of DPPH radical scavenging activity for astaxanthins was 233.33 ± 4.18 μg mL⁻¹. It has been well known that astaxanthins are very unstable under environmental conditions, so the electrospinning technique was used to enhance their stability. In order to fabricate CA nanofibers containing a crude acetone extract of H. pluvialis, various solvent systems and percent loading of the crude acetone extract were studied. The optimal solvent system for fabrication of CA nanofibers was the acetone/dimethylformamide (DMF) system (2 : 1 v/v) with incorporation of 0.25% v/v Tween80, resulting in good morphology of CA nanofibers with av. 420 nm diameter. The loading efficiency (%) of the crude astaxanthins extract was 5% w/w of CA. With regard to the results of the in vivo oxidative stress assay, C. elegans pre-treated with 200 μg mL⁻¹ of the crude extract had a survival percent of 56 after administration of 250 mM of paraquat for 8 h. Under phosphate-buffered saline (pH 7.4) containing 10% v/v acetone, the release of astaxanthins from the CA nanofibers loaded with the crude extract exhibited a prolonged profile. The stability of astaxanthins in electrospun CA nanofibers was examined using the freeze–thaw cycle testing through a DPPH radical scavenging assay. It was found that their stability was significantly different (P < 0.05) after the 12^th freeze–thaw cycle compared with the crude extract.

The astaxanthin derivatives loaded CA fibers enhanced the percentage survival of C. elegans upto 56.17% after adding paraquat and it stability increased with 1.7 times by using DPPH scavenging assay.     

Susceptibility of Neisseria gonorrhoeae to antimicrobial peptides from amphibian skin, dermaseptin, and derivatives

We evaluated the antimicrobial effect of antimicrobial peptides from frog skin belonging to the dermaseptin family against reference and clinical Neisseria gonorrhoeae strains, including penicillin-resistant strains. Dermaseptin S4 exhibited anti-N. gonorrhoeae activity against all strains with MICs ranging between 10 and 100 microg/mL. We then used derivatives of DS4 and determined the anti-N. gonorrhoeae activity of each of analogs. All the derivatives showed antimicrobial activity. Among the different molecules tested, we found that dermaseptins K4S4 (1-16)a and K4S4 (1-28) were the more potent to inhibit N. gonorrhoeae growth with MIC of 10 microg/mL against all strains.     

Antibacterial activity of liposome-entrapped ampicillin in vitro and in vivo in relation to the lipid composition

The antibacterial activity of liposome-entrapped ampicillin against Listeria monocytogenes was investigated in relation to the lipid composition of the liposomes. This was studied in vitro in mouse peritoneal macrophages infected with L. monocytogenes, as well as in vivo in experimental L. monocytogenes infection in mice. Two types of liposomes, a relatively fluid type, consisting of cholesterol-phosphatidylcholine-phosphatidylserine (5:4:1), and a less fluid type, consisting of cholesterol-distearoylphosphatidylcholine-dipalmitoylphosphatidylglyc erol (10:10:1), were used. The uptake of both types of liposomes by macrophages in vitro was similar. However, the rate of intracellular degradation appeared to be dependent on the lipid composition. A correlation was found between the relatively slow degradation of the less fluid liposomes and a delayed intracellular release of the encapsulated ampicillin, as reflected in absent or delayed intracellular killing of L. monocytogenes in vitro. The results obtained in vitro were confirmed by the observations in vivo. Slow degradation of the less fluid liposomes in vivo resulted in a decrease in the therapeutic effect of the antibiotic.     

Enhancement of rabbit protein S anticoagulant cofactor activity in vivo by modulation of the protein S C4B binding protein interaction.

The carboxy-terminal region of protein S has been recently been observed to be involved in the interaction between protein S and C4b-binding protein (Walker, F. J. 1989. J. Biol. Chem. 264:17645-17658). A synthetic peptide, GVQLDLDEAI, corresponding to that region of protein S has been used to investigate the protein S/C4b-binding protein interaction in vitro and in vivo. Rabbit activated protein C possesses species-specific anticoagulant activity for which rabbit protein S functions as a cofactor. In plasma, rabbit protein S is found in complex with C4b-binding protein. GVQLDLDEAI can inhibit this interaction, resulting in enhancement of the anticoagulant activity of rabbit activated protein C. The effect of the peptide can be blocked by the concurrent addition of human or rabbit C4b-binding protein. When infused into rabbits, GVQLDLDEAI was cleared from the circulation with a half-life of 80 min. This is significantly less rapid than the clearance of similarly sized control peptides (half-life of 15 min), but much more than that of bovine protein S, a much larger protein (half-life of 15 h). Plasma samples removed from the rabbits after infusion with GVQLDLDEAI were found to have increased concentrations of free protein S and to show enhanced anticoagulation by rabbit activated protein C ex vivo in a dose-dependent manner. The concentration for half-maximal effect (5 microM) was very similar to that observed in vitro. These results suggest that the formation of a complex between protein S and C4b-binding protein is important in the regulation of protein S activity in vivo, and that modulation of this interaction allows one to influence the anticoagulant activity of the protein C pathway.     

Antibacterial activity of lomefloxacin

Lomefloxacin has marked activity against Gram-negative bacilli including Enterobacteriaceae, non-fermenting strains and Haemophilus influenzae with 98% of all isolates tested having MICs of 0.25 mg/l or less. Sixty-eight per cent of Pseudomonas aeruginosa strains were sensitive to 1 mg/l with a few strains resistant to 8 or 16 mg/l. Gram-positive cocci were more resistant, particularly streptococci, where the MICs vary between 1 and 8 mg/l. Bactericidal activity was similar to inhibitory activity and the effect of increasing serum concentrations and bacterial inocula was minimal. The MIC and MBC were increased in the presence of urine, particularly at an acid pH 5. Comparative MICs showed that lomefloxacin was more active than ofloxacin and pefloxacin, similar to norfloxacin but less active than ciprofloxacin for Gram-negative bacteria but not for Gram-positive cocci. Comparative studies with sensitivity disc concentrations showed that a 5 micrograms disc was more satisfactory than the 10 micrograms disc as the zone sizes were more suitable for routine testing. Solutions of lomefloxacin showed instability in bright sunlight when 52% of activity was lost in 1 h. Similar instability was shown in impregnated discs which lost up to 40% activity in 6 h exposure. Lomefloxacin showed a wide range of activity against Gram-negative bacteria including multiresistant strains and Pseudomonas spp. Gram-positive bacteria were less susceptible, with streptococci more resistant than staphylococci. Lomefloxacin is well absorbed after oral administration giving high blood and urine concentrations and its prolonged half-life means once daily dosing in the treatment of many types of bacterial infection may be possible.