C-Terminal Modification and Multimerization Increase the Efficacy of a Proline-Rich Antimicrobial Peptide.

Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 µm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 µm/8 µg mL-1 ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.

Multimerization of a Proline-Rich Antimicrobial Peptide, Chex-Arg20, Alters Its Mechanism of Interaction with the Escherichia coli Membrane.

A3-APO, a de novo designed branched dimeric proline-rich antimicrobial peptide (PrAMP), is highly effective against a variety of in vivo bacterial infections. We undertook a selective examination of the mechanism for the Gram-negative Escherichia coli bacterial membrane interaction of the monomer (Chex-Arg20), dimer (A3-APO), and tetramer (A3-APO disulfide-linked dimer). All three synthetic peptides were effective at killing E. coli. However, the tetramer was 30-fold more membrane disruptive than the dimer while the monomer showed no membrane activity. Using flow cytometry and high-resolution fluorescent microscopy, it was observed that dimerization and tetramerization of the Chex-Arg20 monomer led to an alteration in the mechanism of action from non-lytic/membrane hyperpolarization to membrane disruption/depolarization. Our findings show that the membrane interaction and permeability of Chex-Arg20 was altered by multimerization.

Susceptibility of intracellular Coxiella burnetii to antimicrobial peptides in mouse fibroblast cells.

Q fever is a zoonotic disease caused by Coxiella burnetii, an obligate intracellular bacterium that resides inside a phagolysosome-like niche. Chronic Q fever is typified by endocarditis, and is treated with multiple antibiotics for at least 18 months. The discovery of clinical C. burnetii isolates resistant to the first-line antibiotic doxycycline, and the problematic nature of chronic Q fever treatment have demonstrated the need for improved treatment regimes. To search for alternative antimicrobial agents, we assessed the effect of 26 antimicrobial peptides (AMPs) on the intracellular growth of C. burnetii in L929 cells at a concentration of 25 µM or their maximal non-cytotoxic concentration. Among the peptides tested, A3-APO, Cath-BF, δ-Hemolysin, Octa-1, P5 and Pleurocidin were able to significantly reduce both the total bacterial cell number and the host cell bacterial burden (average bacterial number per host cell). Combining selected AMPs with Chariot, a non-covalent carrier peptide, did not increase treatment potency when non-cytotoxic concentrations were used, with the exception of P5, which remained active at a concentration of 1.6 µM (1.8 µg/mL). Combining AMPs with each other did not further improve AMP potency, with some treatment combinations increasing the growth rate of C. burnetii by >3-fold. This is the first description of AMP cellular penetration to exhibit inhibitory affect on intracellular C. burnetii growth. These results are the first step in the development of a non-traditional antibiotic treatment for Q fever.

The designer proline-rich antibacterial peptide A3-APO prevents Bacillus anthracis mortality by deactivating bacterial toxins.

Proline-rich antibacterial peptides protect experimental animals from bacterial challenge even if they are unable to kill the microorganisms in vitro. Their major in vivo modes of action are inhibition of bacterial protein folding and immunostimulation. Here we investigated whether the proline-rich antibacterial peptide dimer A3-APO was able to inhibit Bacillus cereus enterotoxin production in vitro and restrict the proliferation of lethal toxin-induced Bacillus anthracis replication in mouse macrophages. After 24 h incubation, peptide A3-APO and its single chain metabolite reduced the amount of properly folded B. cereus diarrhoeal enterotoxin production in a concentration-dependent manner leading to only 10-25% of the original amount of toxin detectable by a conformation-sensitive immunoassay. Likewise, after 4 h incubation, A3-APO restricted the proliferation of B. anthracis in infected macrophages by 40-45% compared to untreated cells both intracellularly and in the extracellular cell culture milieu. Although the peptide had a minimal inhibitory concentration of >512 mg/L against B. anthracis in vitro, in systemic mouse challenge models it improved survival by 20- 37%, exhibiting statistically significant cumulative efficacy when administered at 3x5 mg/kg intraperitoneally or intramuscularly. We hypothesize that the activity in isolated murine macrophages and in vivo is due to deactivation of bacterial toxins. Bacterial protein folding inhibition in synergy with other types of antimicrobial modes offers a remarkable novel strategy in combating resistant or life-threatening infections.

Chemical synthesis and biological evaluation of an antimicrobial peptide gonococcal growth inhibitor.

Gonococcal growth inhibitor 1 (GGI-1) is a 44-residue peptide with potent anti-Legionella activity. It has been isolated from Staphylococcus haemolyticus but, to date, its chemical synthesis has not been reported. Acquisition of this peptide via this means would enable a more detailed examination of its antimicrobial properties. However, its synthesis represents a significant challenge because of two predicted "difficult sequences" within the peptide. Its successful solid-phase assembly is reported in this paper, and was accomplished by use of simple palliative measures including the introduction of a single pseudo-proline isostere in order to counteract on-resin aggregation. The peptide had moderate antimicrobial activity against Escherichia coli but was inactive against another Gram-negative bacterium and two Gram-positive bacteria (Bacillus species). It had significant haemolytic activity, with a H(50) (concentration of peptide that causes 50 % haemolysis) of 20 and 125 µM for two blood samples from different donors. An alternative therapeutic index to that proposed for GGI-1 in a recent publication is proposed.

Synthesis of 1,2,3-triazole linked galactopyranosides and evaluation of cholera toxin inhibition.

We report the synthesis of a series of bivalent 1,2,3-triazole linked galactopyranosides as potential inhibitors of cholera toxin (CT). The inhibitory activity of the bivalent series was examined (ELISA) and the series showed low inhibitory activity (millimolar IC(50)s). Conversely, the monomeric galactotriazole analogues were strong inhibitors of cholera toxin (IC(50) = 71-75 µM).

Potent antimicrobial peptides with selectivity for Bacillus anthracis over human erythrocytes.

In this study, 39 antimicrobial peptides, most with documented low haemolytic activity and potent efficacy against Gram-negative and Gram-positive bacteria, were evaluated for their haemolytic activity against human red blood cells as well as their antimicrobial activity against Escherichia coli, Burkholderia thailandensis, Bacillus globigii and Bacillus anthracis. The majority of the peptides had a minimum inhibitory concentration (MIC) of <10 µM against B. globigii. However, only eight of these (CaLL, Ci-MAM-A24, LLaMA, Ltc2a, OV-5, papillosin, smapspin and smapspin-G) had a MIC<10 µM against B. anthracis. All except one (papillosin) were ineffective at 100 µM against B. thailandensis and none had potent activity against E. coli. Potent activity against B. anthracis was associated with significant haemolytic activity, but the ratio of the concentration of peptide that caused 50% haemolysis to the concentration that inhibited growth of B. anthracis by 50% (the therapeutic index) varied from 0.8 to 34.2. Two peptides (papillosin and Ltc2a) had a therapeutic index >30 and could be considered as candidates for further development for potential medical countermeasures against anthrax. Although B. globigii has often been used as a non-pathogenic simulant for B. anthracis, in this study it was found that the sensitivity of B. globigii to peptides was not a reliable predictor of the sensitivity of B. anthracis to the same peptides.

Analogues of peptide SMAP-29 with comparable antimicrobial potency and reduced cytotoxicity.

SMAP-29 (sheep myeloid antimicrobial peptide-29) is a peptide with potent antibacterial properties. However, it is also highly cytotoxic both to human red blood cells (hRBCs) and human embryonic kidney (HEK) cells. In this study, some of the amino acids of SMAP-29 were changed in an attempt to reduce haemolytic activity whilst maintaining high antibacterial efficacy. These analogues, plus other analogues described in the literature with potent antimicrobial activity against Gram-positive bacteria coupled with no or low haemolytic activity, were evaluated for their cytotoxicity (hRBCs and HEK cells) as well as antimicrobial efficacy against two Gram-positive (Bacillus anthracis and Bacillus globigii) and two Gram-negative bacteria (Escherichia coli and Burkholderia thailandensis). The analogues previously described in the literature were found to have low antibacterial and haemolytic activity. Two of the designed analogues had comparable antibacterial efficacy with SMAP-29 against B. anthracis but reduced haemolytic activity and therefore had a therapeutic index that was enhanced 2.3-2.6-fold over that of SMAP-29.

Characterisation and evaluation of synthetic antimicrobial peptides against Bacillus globigii, Bacillus anthracis and Burkholderia thailandensis.

Antimicrobial peptides (AMPs) are produced by all forms of living organisms and represent a novel class of antibiotics to treat infectious diseases. In this study, 29 AMPs of varying length and characteristics were synthesised chemically and were evaluated for their ability to inhibit the growth of Bacillus globigii, Bacillus anthracis and Burkholderia thailandensis. Amongst the peptides tested, sheep myeloid antimicrobial peptide-29 (SMAP-29) was the most potent, inhibiting both B. globigii and B. anthracis at submicromolar concentrations. However, SMAP-29 was less effective against B. thailandensis (minimum inhibitory concentration of 71 microM). Haemolytic activity and cytotoxicity were determined using human blood cells and human embryonic kidney 293S cells, respectively. Most of the peptides tested showed varying degrees of haemolytic activity and cytotoxicity, with SMAP-29 being highly haemolytic and cytotoxic under the conditions tested. Nevertheless, strategies to reduce toxicity whilst maintaining high antimicrobial activity are worth pursuing in light of the results obtained.

The CMG2 ELISA for evaluating inhibitors of the binding of anthrax toxin protective antigen to its receptor.

INTRODUCTION: Anthrax toxin comprises a protective antigen (PA) of MW 83 kDa, a lethal factor (LF) and an edema factor (EF). Upon binding to its receptor on cell surfaces, PA(83) is enzymatically cleaved to a 63 kDa product (PA(63)), followed by binding of LF or EF, receptor-mediated internalisation of these factors, and production of their toxic effects. The high-affinity binding of PA(83) to its receptor is essential for the intoxication process. To study the interaction between the PA and its receptor, and inhibition of the binding, an enzyme-linked immunosorbent assay (ELISA) was developed. METHODS: One of the two known anthrax toxin receptors (capillary morphogenesis factor 2; CMG2) was adsorbed onto wells of a 96-well plate. Either PA(83) or PA(63) was then added to the receptor-coated wells, followed by sequential addition of anti-PA antibody, anti-species antibody-enzyme conjugate, and enzyme substrate at appropriate time intervals. RESULTS: Best results were obtained by overnight incubation of CMG2 in PBS at 4 degrees C. CMG2 was used at 1 microg/ml because of the cost of the commercial product. The rate of change of absorbance was low, and was measured over 3 h to obtain accurate results. The assay results increased almost linearly with CMG2 concentration to 10 microg/ml. PA(83) was also used at 1 microg/ml, but the assay values reached a plateau at approx. 10 microg/ml. Binding was divalent cation-dependent, almost irreversible, and free CMG2 was a potent inhibitor of binding (I(50) in the nM range). Binding of PA(63) was similar to that of PA(83). DISCUSSION: The high-affinity binding and divalent cation dependence confirm the validity of the assay as a model for toxin-receptor binding in vivo and as a means of evaluating toxin-receptor binding and inhibitors of the binding. Attempts to use crude lysates of J774A.1 cells or von Willebrand factor as an alternative source of anthrax toxin receptor were not successful.

Biofunctionalized surfactant mesophases as polyvalent inhibitors of cholera toxin.

The cubic lyotropic mesophase composed of the ganglioside G(M1) and the synthetic surfactant phytantriol has been employed as a scaffold to prepare a polyvalent inhibitor of cholera toxin (CT). Surfactant mixtures containing up to 20% (w/w) G(M1)/phytantriol afforded a hydrated insoluble gel-like material, which retained an inverse cubic phase (Q) structure in excess water and at 22 degrees C, as confirmed using small-angle X-ray scattering. The G(M1)-functionalized mesophases bind up to 98.8% of CT from solution containing 100 ng/mL of CT with a dissociation constant (K(d)) of 67-73 nM. The estimated IC50 values for the mesophase systems were 0.1-0.27 microM. The inhibitory effect of the mesophases may be enhanced through the high internal surface area of the inverse cubic phase in addition to the optimal self-orientation of G(M1) ligand to match the distribution of binding sites on the toxin surface. As a result, polyvalent inhibitor materials manufactured using these mesophase structures do not require precise control of ligand distribution, which offers advantages with respect to complexity of synthesis and formulation when compared to more rigid and conformationally restricted materials. This approach provides a route to a unique class of polyvalent inhibitors, which should be broadly applicable to a range of bacterial and plant toxins.