Improvement of cyclic decapeptides against plant pathogenic bacteria using a combinatorial chemistry approach

Cyclic decapeptides were developed based on the previously reported peptide c(LysLeuLysLeuLysPheLysLeuLysGln). These compounds were active against the economically important plant pathogenic bacteria Erwinia amylovora, Pseudomonas syringae and Xanthomonas vesicatoria. A library of 56 cyclic decapeptides was prepared and screened for antibacterial activity and eukaryotic cytotoxicity, and led to the identification of peptides with improved minimum inhibitory concentration (MIC) against P. syringae (3.1-6.2 microM) and X. vesicatoria (1.6-3.1 microM). Notably, peptides active against E. amylovora (MIC of 12.5-25 microM) were found, constituting the first report of cyclic peptides with activity towards this bacteria. A second library based on the structure c(X(1)X(2)X(3)X(4)LysPheLysLysLeuGln) with X being Lys or Leu yielded peptides with optimized activity profiles. The activity against E. amylovora was further improved (MIC of 6.2-12.5 microM) and the best peptides displayed a low eukaryotic cytotoxicity at concentrations 30-120 times higher than the MIC values. A design of experiments permitted to define rules for high antibacterial activity and low cytotoxicity, being the main rule X(2) not equal X(3), and the secondary rule X(4)=Lys. The best analogs can be considered as good candidates for the development of effective antibacterial agents for use in plant protection.     

Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2

Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a membrane-mimetic environment, the peptide exists in an amphipathic alpha-helical conformation. Analogs of the peptide with increased cationicity and alpha-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic face of the alpha-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [D-Lys3, D-Lys10, D-Lys14]pseudin-2 showed potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 microM against several antibiotic-resistant strains of Escherichia coli) but very low hemolytic activity (HC50>500 microM) and cytolytic activity against L929 fibroblasts (LC50=215 microM). Increasing the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human erythrocytes. Time-kill studies demonstrated that the analog [Lys3, Lys10, Lys14, Lys21]pseudin-2 at a concentration of 1 x MIC was bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the alpha-helix by L-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.     

Design and mechanism of action of a novel bacteria-selective antimicrobial peptide from the cell-penetrating peptide Pep-1

Here, we report the successful design of a novel bacteria-selective antimicrobial peptide, Pep-1-K (KKTWWKTWWTKWSQPKKKRKV). Pep-1-K was designed by replacing Glu-2, Glu-6, and Glu-11 in the cell-penetrating peptide Pep-1 with Lys. Pep-1-K showed strong antibacterial activity against reference strains (MIC = 1-2 microM) of Gram-positive and Gram-negative bacteria as well as against clinical isolates (MIC = 1-8 microM) of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. In contrast, Pep-1-K did not cause hemolysis of human erythrocytes even at 200 microM. These results indicate that Pep-1-K may be a good candidate for antimicrobial drug development, especially as a topical agent against antibiotic-resistant microorganisms. Tryptophan fluorescence studies indicated that the lack of hemolytic activity of Pep-1-K correlated with its weak ability to penetrate zwitterionic phosphatidylcholine/cholesterol (10:1, w/w) vesicles, which mimic eukaryotic membranes. Furthermore, Pep-1-K caused little or no dye leakage from negatively charged phosphatidylethanolamine/phosphatidylglycerol (7:3, w/w) vesicles, which mimic bacterial membranes but had a potent ability to cause depolarization of the cytoplasmic membrane potential of intact S. aureus cells. These results suggested that Pep-1-K kills microorganisms by not the membrane-disrupting mode but the formation of small channels that permit transit of ions or protons but not molecules as large as calcein.     

Peptides with antimicrobial and anti-inflammatory activities that have therapeutic potential for treatment of acne vulgaris

The pathogenesis of acne vulgaris is multifactorial involving infection of the pilosebaceous unit with Propionibacterium acnes and a cytokine-mediated inflammatory response. Five frog skin-derived antimicrobial peptides ([D4k]ascaphin-8, [G4K]XT-7, [T5k]temporin-DRa, brevinin-2GU, and B2RP-ERa), chosen for their low hemolytic activity against human erythrocytes, were assessed for their effects on the growth of clinical isolates of P. acnes and on the release of pro-inflammatory and anti-inflammatory cytokines from peripheral blood mononuclear (PBM) cells. All peptides inhibited the growth of P. acnes with the highest potency exhibited by [D4k]ascaphin-8 (minimum inhibitory concentration, MIC=3-12.5 μM). Release of TNF-α from concanavalin A (ConA)-stimulated PBM cells was significantly reduced by [D4k]ascaphin-8, [G4K]XT-7, brevinin-2GU, and B2RP-ERa (1 and 20 μg/ml) and by [T5k]temporin-DRa (20 μg/ml). Release of IFN-γ from unstimulated PBM cells was significantly reduced by [D4k]ascaphin-8 and brevinin-2GU (1 and 20 μg/ml). No peptide showed significant effects on Il-17 release. Release of the anti-inflammatory cytokines TGF-β, IL-4, and IL-10 from both unstimulated and ConA-treated PBM cells was significantly increased by [T5k]temporin-DRa and B2RP-ERa (1 and 20μg/ml). The potent activities of [D4k]ascaphin-8 and [T5k]temporin-DRa in inhibiting the growth of P. acnes and the release of pro-inflammatory cytokines, and in stimulating the release of anti-inflammatory cytokines suggest a possible therapeutic role in the treatment of acne vulgaris.     

De novo designed cyclic cationic peptides as inhibitors of plant pathogenic bacteria

Head-to-tail cyclic peptides of 4-10 residues consisting of alternating hydrophilic (Lys) and hydrophobic (Leu and Phe) amino acids were synthesized and tested against the economically important plant pathogenic bacteria Erwinia amylovora, Xanthomonas vesicatoria and Pseudomonas syringae. The antibacterial activity, evaluated as the minimal inhibitory concentration (MIC), the cytotoxicity against human red blood cells and stability towards protease degradation were determined. The influence of cyclization, ring size, and replacement of l-Phe with d-Phe on antibacterial and hemolytic activities was studied and correlated with the degree of structuring and hydrophobicity. Our results showed that linear peptides were inactive against the three bacteria tested. Cyclic peptides were active only toward X. vesicatoria and P. syringae, being c(KLKLKFKLKQ) (BPC10L) the most active peptide with MIC values of 6.25 and 12.5 microM, respectively. The improved antibacterial activity of cyclic peptides compared to their linear counterparts was associated to an increase of the hydrophobicity, represented as RP-HPLC retention time (t(R)), and secondary structure content which are related to an enhanced amphipathicity. A decrease of antibacterial and hemolytic activities was observed when a d-Phe was introduced into the cyclic sequences, which was attributed to their low amphipathicity as shown by their low secondary structure content and low t(R). The small size, simple structure, bactericidal effect, and stability to protease degradation of the best peptides make them potential candidates for the development of effective antibacterial agents for use in plant protection.     

Structure and fungicidal activity of a synthetic antimicrobial peptide, P18, and its truncated peptides

P18 (KWKLFKKIPKFLHLAKKF-NH2) is an antimicrobial peptide designed from a cecropin A-magainin 2 hybrid that has potent antibacterial activity without hemolytic activity against human erythrocytes. In this study, P18 displayed potent fungicidal activity (MIC: 12.5 approximately 25 microM) against pathogenic fungi, Candida albicans, Trichosporon beigelii, Aspergillus flavus and Fusarium oxyspovrum. The central Pro9 residue and the entire sequence of P18 are essential for its full fungicidal activity. Circular dichroism analysis suggested that the higher alpha-helical content of the peptides did not correlate with the stronger fungicidal activity.     

Antimicrobial properties of the frog skin peptide, ranatuerin-1 and its [Lys-8]-substituted analog

The predicted conformation of ranatuerin-1 (SMLSVLKNLG(10)KVGLGFVACK(20)INK QC), an antimicrobial peptide first isolated from the skin of the bullfrog Rana catesbeiana, comprises three structural domains: alpha-helix (residues 1-8), beta-sheet (residues 11-16) and beta-turn (residues 20-25). Circular dichroism studies confirm significant alpha-helical character in 50% trifluoroethanol. Replacement of Cys-19 and Cys-25 by serine resulted only in decreased antimicrobial potency but deletion of either the cyclic heptapeptide region [residues (19-25)] or the N-terminal domain [residues (1-8)] produced inactive analogs. Substitution of the glycine residues in the central domain of the [Ser-19, Ser-25] analog by lysine produced inactive peptides despite increased alpha-helical content and cationicity. The substitution Asn-8-->Lys gave a ranatuerin-1 analog with increased alpha-helicity and cationicity and increased potency against a range of Gram-positive and Gram-negative bacteria and against C. albicans but only a small increase (21%) in hemolytic activity. In contrast, increasing alpha-helicity and hydrophobicity by the substitution Asn-22-->Ala resulted in a 3.5-fold increase in hemolytic activity. Effects on antimicrobial potencies of substitutions of neutral amino acids at positions 4, 18, 22, and 24 by lysine were less marked. Strains of pathogenic E. coli from different groups showed varying degrees of sensitivity to ranatuerin-1 (MIC between 5 and 40 microM) but [Lys-8] ranatuerin-1 showed increased potency (between 2- and 8-fold; P < 0.01) against all strains. The data demonstrate that [Lys-8] ranatuerin-1 shows potential as a candidate for drug development.     

3-[Benzimidazo- and 3-[benzothiadiazoleimidazo-(1,2-c)quinazolin-5-yl]-2H-chromene-2-ones as potent antimicrobial agents

A series of 3-[benzimidazo(1,2-c)quinazolin-5-yl]-2H-chromene-2-one (6a-6f) and 3-[benzothiadiazole- imidazo(1,2-c)quinazolin-5-yl]-2H-chromene-2-one derivatives (7a-7f) that incorporate a variety of substituents at the 6- and/or 8-positions of the coumarin moieties have been synthesized utilizing cellulose sulfuric acid as an efficient catalyst under both conventional heating and microwave irradiation procedures. These analogs were evaluated for their antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogenes (Gram-positive bacteria), Escherichia Coli, Klebsiella pneumonia, Salmonella typhimurium (Gram-negative bacteria), and Aspergillus niger, Candida albicans, and Aspergillus flavus (Fungi). Two analogs, 6c (a 6,8-dichloro analog, MIC([SA]) = 2.5 μg/mL; MIC([ST]) = 2.5 μg/mL) and 7d (a 6,8-dibromo analog, MIC([ST]) = 2.5 μg/mL) were identified as potent antibacterial agents, and two analogs, 6b (a 6-bromo analog, MIC([AF]) = 10 μg/mL) and 6d (a 6,8-dibromo analog, MIC([AF]) = 15 μg/mL; MIC([CA]) = 15μg/mL), were identified as potent antifungal agents. Based on the MIC data, analogs 6b, 6c, 6d, and 7d were identified as the most potent antimicrobial agents in the series.     

Antimicrobial and cytolytic properties of the frog skin peptide, kassinatuerin-1 and its L- and D-lysine-substituted derivatives

Kassinatuerin-1, a 21-amino-acid C-terminally alpha-amidated peptide first isolated from the skin of the African frog Kassina senegalensis, adopts an amphipathic alpha-helical conformation in a membrane-mimetic solvent (50% trifluoroethanol) and shows broad-spectrum antimicrobial activity. However, its therapeutic potential is limited by its relatively high cytolytic activity against mammalian cells. The antimicrobial and cytolytic properties of a peptide are determined by an interaction between cationicity, hydrophobicity, alpha-helicity and amphipathicity. Replacement of the C-terminal alpha-amide group in kassinatuerin-1 by carboxylic acid decreased both cationicity and alpha-helicity, resulting in an analog with decreased potency against Escherichia coli (4-fold) and Staphylococcus aureus (16-fold). Low cytolytic activities against human erythrocytes (LD50>400 microM) and L929 fibroblasts (LD50=105 microM) were also observed. Increasing cationicity, while maintaining amphipathic alpha-helical character, by progressively substituting Gly7, Ser18, and Asp19 on the hydrophilic face of the alpha-helix with L-lysine, increased antimicrobial potency against S. aureus and Candida albicans (up to 4-fold) but also increased hemolytic and cytolytic activities. In contrast, analogs with d-lysine at positions 7, 18 and 19 retained activity against Gram-negative bacteria but displayed reduced hemolytic and cytolytic activities. For example, the carboxylic acid derivative of [D-Lys7, D-Lys18, D-Lys19]kassinatuerin-1 was active (minimum inhibitory concentration (MIC)=6-12.5 microM) against a range of strongly antibiotic-resistant strains of E. coli but showed no detectable hemolytic activity at 400 microM and was 4-fold less cytolyic than kassinatuerin-1. However, the reduction in alpha-helicity produced by the D-amino acid substitutions resulted in analogs with reduced potencies against Gram-positive bacteria and against C. albicans.     

Conformationally restricted analogs of deoxynegamycin

Deoxynegamycin (1b) is a protein synthesis inhibitor with activity against Gram-negative (GN) bacteria. A series of conformationally restricted analogs were synthesized to probe its bioactive conformation. Indeed, some of the constrained analogs were found to be equal or better than deoxynegamycin in protein synthesis assay (1b, IC(50)=8.2 microM; 44, IC(50)=6.6 microM; 35e(2), IC(50)=1 microM). However, deoxynegamycin had the best in vitro whole cell antibacterial activity (Escherichia coli, MIC=4-16 microg/mL; Klebsiella pneumoniae, MIC=8 microg/mL) suggesting that other factors such as permeation may also be contributing to the overall whole cell activity. A new finding is that deoxynegamycin is efficacious in an E. coli murine septicemia model (ED(50)=4.8 mg/kg), providing further evidence of the favorable in vivo properties of this class of molecules.     

Synthesis and antibacterial activity of novel and potent DNA gyrase inhibitors with azole ring

The 4-piperidyl moiety and the pyrazole ring in 1-(3-chlorophenyl)-5-(4-phenoxyphenyl)-3-(4-piperidyl)pyrazole 2, which has previously shown improved DNA gyrase inhibition and target-related antibacterial activity, were transformed to other groups and the in vitro antibacterial activity of the synthesized compounds was evaluated. The selected pyrazole, oxazole and imidazole derivatives showed moderate inhibition against DNA gyrase and topoisomerase IV with similar IC(50) values (IC(50)=9.4-25 microg/mL). In addition, many of the pyrazole, oxazole and imidazole derivatives synthesized in this study exhibited potent antibacterial activity against quinolone-resistant clinical isolates and coumarin-resistant laboratory isolates of Gram-positive bacteria with minimal inhibitory concentration values equivalent to those against susceptible strains.     

Conformational analysis of the broad-spectrum antibacterial peptide, ranatuerin-2CSa: identification of a full length helix-turn-helix motif

Design of clinically valuable antibacterial agents based upon naturally occurring peptides requires the use of spectroscopic methods, particularly NMR, to determine the three-dimensional structure of the native peptide so that analogues with improved therapeutic properties can be made. Ranatuerin-2CSa (GILSSFKGVAKGVAKDLAG KLLETLKCKITGC), first isolated from skin secretions of the Cascades frog, Rana cascadae, represents a promising candidate for drug development. The peptide shows potent growth inhibitory activity against Escherichia coli (MIC=5 microM) and Staphylococcus aureus (MIC=10 microM) but displays haemolytic activity against human erythrocytes (LC(50)=160 microM). The solution structure of ranatuerin-2CSa was investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, the peptide lacks secondary structure but, in a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O solvent mixture, the structure is characterised by a full length helix-turn-helix conformation between residues I(2)-L(21), L(22)-L(25) and K(26)-T(30) respectively. This structural information will facilitate the design of novel therapeutic agents based upon the ranatuerin-2CSa structure with improved antimicrobial potencies but decreased cytolytic activities against mammalian cells.     

New antibacterial peptide derived from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.     

Screening of Indonesian peat soil bacteria producing antimicrobial compounds

The development and world-wide spread of multidrug-resistant (MDR) bacteria have a high concern in the medicine, especially the extended-spectrum of beta-lactamase (ESBL) producing Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). There are currently very limited effective antibiotics to treat infections caused by MDR bacteria. Peat-soil is a unique environment in which bacteria have to compete each other to survive, for instance, by producing antimicrobial substances. This study aimed to isolate bacteria from peat soils from South Kalimantan Indonesia, which capable of inhibiting the growth of Gram-positive and Gram-negative bacteria. Isolates from peat soil were grown and identified phenotypically. The cell-free supernatant was obtained from broth culture by centrifugation and was tested by agar well-diffusion technique against non ESBL-producing E. coli ATCC 25922, ESBL-producing E. coli ATCC 35218, methicillin susceptible Staphylococcus aureus (MSSA) ATCC 29,213 and MRSA ATCC 43300. Putative antimicrobial compounds were separated using SDS-PAGE electrophoresis and purified using electroelution method. Antimicrobial properties of the purified compounds were confirmed by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). In total 28 isolated colonies were recovered; three (25PS, 26PS, and 27PS) isolates produced proteins with strong antimicrobial activities against both reference strains. The substance of proteins from three isolates exerted strong antimicrobial activity against ESBL-producing E. coli ATCC 35,218 (MIC = 2,80 µg/mL (25PS), 3,76 µg/mL (26PS), and 2,41 µg/mL (27PS), and MRSA ATCC 43,300 (MIC = 4,20 µg/mL (25PS), 5,65 µg/mL (26PS), and 3,62 µg/mL (27PS), and also had the ability bactericidal properties against the reference strains. There were isolates from Indonesian peat which were potentials sources of new antimicrobials.     

Role of Phenylalanine and Valine10 Residues in the Antimicrobial Activity and Cytotoxicity of Piscidin-1

Piscidin-1 (Pis-1) is a linear antibacterial peptide derived from mast cells of aquacultured hybrid striped bass that comprises 22 amino acids with a phenylalanine-rich amino-terminus. Pis-1 exhibits potent antibacterial activity against pathogens but is not selective for distinguishing between bacterial and mammalian cells. To determine the key residues for its antibacterial activity and those for its cytotoxicity, we investigated the role of each Phe residue near the N-terminus as well as the Val¹⁰ residue located near the boundary of the hydrophobic and hydrophilic sectors of the helical wheel diagram. Fluorescence dye leakage and tryptophan fluorescence experiments were used to study peptide-lipid interactions, showing comparable depths of insertion of substituted peptides in different membranes. Phe² was found to be the most deeply inserted phenylalanine in both bacterial- and mammalian-mimic membranes. Each Phe was substituted with Ala or Lys to investigate its functional role. Phe² plays key roles in the cytotoxicity as well as the antibacterial activities of Pis-1, and Phe⁶ is essential for the antibacterial activities of Pis-1. We also designed and synthesized a piscidin analog, Pis-V10K, in which Lys was substituted for Val¹⁰, resulting in an elevated amphipathic α-helical structure. Pis-V10K showed similar antibacterial activity (average minimum inhibitory concentration (MIC)  = 1.6 µM) to Pis-1 (average MIC  = 1.5 µM). However, it exhibited much lower cytotoxicity than Pis-1. Lys¹⁰-substituted analogs, Pis-F1K/V10K, Pis-F2K/V10K, and Pis-F6K/V10K in which Lys was substituted for Phe retained antibacterial activity toward standard and drug-resistant bacterial strains with novel bacterial cell selectivity. They exert anti-inflammatory activities via inhibition of nitric oxide production, TNF-α secretion, and MIP-1 and MIP-2 production. They may disrupt the binding of LPS to toll-like receptors, eventually suppressing MAPKs-mediated signaling pathways. These peptides may be good candidates for the development of peptide antibiotics with potent antibacterial activity but without cytotoxicity.     

King cobra peptide OH-CATH30 as a potential candidate drug through clinic drug-resistant isolates

Cationic antimicrobial peptides(AMPs) are considered as important candidate therapeutic agents, which exert potent microbicidal properties against bacteria, fungi and some viruses. Based on our previous findings king cobra cathelicidin(OH-CATH) is a 34-amino acid peptide that exerts strong antibacterial and weak hemolytic activity. The aim of this research is to evaluate the efficacy of both OH-CATH30 and its analog D-OH-CATH30 against clinical isolates comparing with routinely utilized antibiotics in vitro.In this study, 584 clinical isolates were tested(spanning 2013-2016) and the efficacy of the candidate peptides and antibiotics were determined by a broth microdilution method according to the CLSI guidelines. Among the 584 clinical isolates, 85% were susceptible to OH-CATH30 and its analogs. Both L-and D-OH-CATH30 showed higher efficacy against(toward) Gram-positive bacteria and stronger antibacterial activity against nearly all Gram-negative bacteria tested compare with antibiotics. The highest bactericidal activity was detected against Acinetobacter spp., including multi-drug-resistant Acinetobacter baumannii(MRAB)and methicillin-resistant Staphylococcus aureus(MRSA). The overall efficacy of OH-CATH30 and its analogs was higher than that of the 9 routinely used antibiotics. OH-CATH30 is a promising candidate drug for the treatment of a wide variety of bacterial infections which are resistant to many routinely used antimicrobial agents.     

Structure--activity study of the antibacterial peptide fallaxin

Fallaxin is a 25-mer antibacterial peptide amide, which was recently isolated from the West Indian mountain chicken frog Leptodactylus fallax. Fallaxin has been shown to inhibit the growth of several Gram-negative bacteria including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Here, we report a structure-activity study of fallaxin based on 65 analogs, including a complete alanine scan and a full set of N- and C-terminal truncated analogs. The fallaxin analogs were tested for hemolytic activity and antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate resistant S. aureus, (VISA), methicillin-susceptible S. aureus (MSSA), E. coli, K. pneumoniae, and P. aeruginosa. We identified several analogs, which showed improved antibacterial activity compared to fallaxin. Our best candidate was FA12, which displayed MIC values of 3.12, 25, 25, and 50 muM against E. coli, K. pneumoniae, MSSA, and VISA, respectively. Furthermore, we correlated the antibacterial activity with various structural parameters such as charge, hydrophobicity H, mean hydrophobic moment mu(H), and alpha-helicity. We were able to group the active and inactive analogs according to mean hydrophobicity H and mean hydrophobic moment mu(H). Far-UV CD-spectroscopy experiments on fallaxin and several analogs in buffer, in TFE, and in membrane mimetic environments (small unilamellar vesicles) indicated that a coiled-coil conformation could be an important structural trait for antibacterial activity. This study provides data that support fallaxin analogs as promising lead structures in the development of new antibacterial agents.     

Synthesis,micellisation and interaction of novel quaternary ammonium compounds derived from l-Phenylalanine with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine as model membrane in relation to their antibacterial activity,and their selectivity over human red blood cells

A series of quaternary ammonium compounds (QUATS) derived from l-Phenylalanine have been synthesized and their antibacterial efficiencies were determined against various strains of Gram-positive and Gram-negative bacteria. The antibacterial activity increased with increasing chain length, exhibiting a cut-off effect at C₁₄ for Gram-positive and C₁₂ for Gram-negative bacteria. The l-Phenylalanine QUATS displayed enhanced antibacterial properties with a higher cut-off point compared to their corresponding l-Phenylalanine ester hydrochlorides. The CMC was correlated with the MIC, inferring that micellar activity contributes to the cut-off effect in antibacterial activity. The hemolytic activities (HC₅₀) of the QUATS against human red blood cells were also determined to illustrate the selectivity of these QUATS for bacterial over mammalian cells. In general, the MIC was lower than the HC₅₀, and assessment of the micellar contribution to the antibacterial and hemolytic evaluation in TBS as a common medium confirmed that these QUATS can act as antibacterial, yet non-toxic molecules at their monomer concentrations. The interaction of the QUATS with the phospholipid vesicles (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) in the presence of 1-anilino-8-naphthalene sulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescence probes showed that the presence of the quaternary ammonium moiety causes an increase in hydrophobic interactions, thus causing an increase in antibacterial activity.     

Structure-activity relationship study of antimicrobial dermaseptin S4 showing the consequences of peptide oligomerization on selective cytotoxicity

To understand how peptide organization in aqueous solution might affect the activity of antimicrobial peptides, the potency of various dermaseptin S4 analogs was assessed against human red blood cells (RBC), protozoa, and several Gram-negative bacteria. Dermaseptin S4 had weak antibacterial activity but potent hemolytic or antiprotozoan effects. K(4)K(20)-S4 was 2-3-fold more potent against protozoa and RBC, yet K(4)K(20)-S4 was more potent by 2 orders of magnitude against bacteria. K(4)-S4 had similar behavior as K(4)K(20)-S4, but K(20)-S4 and analogous negative charge substitutions were as active as dermaseptin S4 or had reduced activity. Binding experiments suggested that potency enhancement was not the result of increased affinity to target cells. In contrast, potency correlated well with aggregation properties. Fluorescence studies indicated that K(20)-S4 and all negative charge substitutions were as aggregated as dermaseptin S4, whereas K(4)-S4 and K(4)K(20)-S4 were clearly less aggregated. Overall, the data indicated that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptide's oligomerization in solution and, hence, for its limited spectrum of action. Moreover, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or RBC implied that aggregation can have dramatic consequences on antibacterial activity. Based on these results, we tested the feasibility of selectivity reversal in the activity of dermaseptin S4. Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge affected dramatically the peptide's activity and resulted in various analogs that displayed potent antibacterial activity but reduced hemolytic activity. Among these, maximal antibacterial activity was displayed by a 15-mer version that was more potent by 2 orders of magnitude compared with native dermaseptin S4. These results emphasize the notion that peptide-based antibiotics represent a highly modular synthetic antimicrobial system and provide indications of how the peptide's physico-chemical properties affect potency and selectivity.     

Azaphilones from the Fungus Penicillium multicolor LZUC-S2 and Their Antibacterial Activity

Two new azaphilones, penimultiones A and B, together with seven known analogs were isolated from the culture of Penicillium multicolor LZUC-S2. Their structures were elucidated by detailed spectroscopic data analysis and chemical transformation. Penimultiones A and B belong to a rare class of azaphilones possessing a 1,3-dioxolane moiety. In addition, all compounds were evaluated for their antibacterial activity against five clinically bacterial strains in vitro, and three compounds showed potent antibacterial activity with minimum inhibitory concentration (MIC) values ranging from 12.5 to 50.0 μg/mL.