Structural basis for the enhanced activity of cyclic antimicrobial peptides:The case of BPC194

AbstractWe report the molecular basis for the differences in activity of cyclic and linear antimicrobial peptides. We iteratively performed atomistic molecular dynamics simulations and biophysical measurements to probe the interaction of a cyclic antimicrobial peptide and its inactive linear analogue with model membranes. We establish that, relative to the linear peptide, the cyclic one binds stronger to negatively charged membranes. We show that only the cyclic peptide folds at the membrane interface and adopts a β-sheet structure characterised by two turns. Subsequently, the cyclic peptide penetrates deeper into the bilayer while the linear peptide remains essentially at the surface. Finally, based on our comparative study, we propose a model characterising the mode of action of cyclic antimicrobial peptides. The results provide a chemical rationale for enhanced activity in certain cyclic antimicrobial peptides and can be used as a guideline for design of novel antimicrobial peptides

Reflections upon separability and distillability

We present an abstract formulation of the so-called Innsbruck-Hannover programme that investigates quantum correlations and entanglement in terms of convex sets. We present a unified description of optimal decompositions of quantum states and the optimization of witness operators that detect whether a given state belongs to a given convex set. We illustrate the abstract formulation with several examples, and discuss relations between optimal entanglement witnesses and n-copy non-distillable states with non-positive partial transpose.Comment: 12 pages, 7 figures, proceedings of the ESF QIT Conference Gdansk, July 2001, submitted to special issue of J. Mod. Op

Rational Design of Cyclic Antimicrobial Peptides Based on BPC194 and BPC198

A strategy for the design of antimicrobial cyclic peptides derived from the lead compounds c(KKLKKFKKLQ) (BPC194) and c(KLKKKFKKLQ) (BPC198) is reported. First, the secondary β-structure of BPC194 and BPC198 was analyzed by carrying out molecular dynamics (MD) simulations. Then, based on the sequence pattern and the β-structure of BPC194 or BPC198, fifteen analogues were designed and synthesized on solid-phase. The best peptides (BPC490, BPC918, and BPC924) showed minimum inhibitory concentration (MIC) values <6.2 μM against Pseudomonas syringae pv. syringae and Xanthomonas axonopodis pv. vesicatoria, and an MIC value of 12.5 to 25 μM against Erwinia amylovora, being as active as BPC194 and BPC198. Interestingly, these three analogues followed the structural pattern defined from the MD simulations of the parent peptides. Thus, BPC490 maintained the parallel alignment of the hydrophilic pairs K1–K8, K2–K7, and K4–K5, whereas BPC918 and BPC924 included the two hydrophilic interactions K3–Q10 and K5–K8. In short, MD simulations have proved to be very useful for ascertaining the structural features of cyclic peptides that are crucial for their biological activity. Such approaches could be further employed for the development of new antibacterial cyclic peptides

Tryptophan-Containing Cyclic Decapeptides with Activity against Plant Pathogenic Bacteria

A library of 66 cyclic decapeptides incorporating a Trp residue was synthesized on solid phase and screened against the phytopathogenic bacteria Pseudomonas syringae pv. syringae, Xanthomonas axonopodis pv. vesicatoria, and Erwinia amylovora. The hemolytic activity of these peptides was also evaluated. The results obtained were compared with those of a collection of Phe analogues previously reported. The analysis of the data showed that the presence of the Trp improved the antibacterial activity against these three pathogens. In particular, 40 to 46 Trp analogues displayed lower minimum inhibitory concentration (MIC) values than their corresponding Phe counterparts. Interestingly, 26 Trp-containing sequences exhibited MIC of 0.8 to 3.1 μM against X. axonopodis pv. vesicatoria, 21 peptides MIC of 1.6 to 6.2 μM against P. syringae pv. syringae and six peptides MIC of 6.2 to 12.5 μM against E. amylovora. Regarding the hemolysis, in general, Trp derivatives displayed a percentage of hemolysis comparable to that of their Phe analogues. Notably, 49 Trp-containing cyclic peptides showed a hemolysis ≤ 20% at 125 μM. The peptides with the best biological activity profile were c(LKKKLWKKLQ) (BPC086W) and c(LKKKKWLLKQ) (BPC108W), which displayed MIC values ranging from 0.8 to 12.5 μM and a hemolysis ≤ 8% at 125 μM. Therefore, it is evident that these Trp sequences constitute promising candidates for the development of new agents for use in plant protection

Simultaneous pore formation and fusion activity of BPC194.

<p>A: The normalized concentration of dextran inside the liposomes, C_av, (filled circles) and the normalized intensity of membrane-associated DiD per liposome (empty squares) at different P/L ratios. B: Confocal images of the lipid vesicles in the DiD and dextran detection channel at three different P/L ratios; α, P/L = 0; β, P/L = 0.1; and γ, P/L = 0.3. C: Positive-FRET upon peptide addition. The emission of Rhodamine increases due to vesicle fusion. Inset: Controls done with the ‘inactive’ linear analog of BPC194, that is, BPC193 at the same peptide concentrations. D: Negative-FRET upon peptide addition. The emission of NBD increases due to a decrease in FRET efficiency as a result of vesicle fusion. E. Quantification of fusion at different P/L ratios and at two different lipid compositions, 125 µM (full circles) and 250 µM (empty squares). D. Representative cryo-TEM micrographs of DOPG vesicles without peptide (control) and with BPC194 or the linear analog BPC193.</p

Overview and statistics of the MD simulations.

<p>The percentage of lipids in the contacting monolayers which, during the simulation, tilt by more than >85° or splay by more than >170° is indicated. The standard error of the average is obtained from the standard deviation between all five simulations. The simulation length and formation of pores is also indicated.</p

Molecular view of the sequence of events of the leaky fusogenic action of cyclic peptides.

<p>A. Initial simulation setup with peptides placed between two bilayers. B. Bridging of proximal leaflets of the two bilayers by BPC194. C. Lipid bulging caused by the action of peptides associated with the bilayers. D. Pre-stalk intermediate accompanied by disordered toroidal pore. E. Close-up of the bridging peptides. F. Close-up of the stalk-pore complex. G–J. Splaying of a lipid during the course of a simulation. The peptides are depicted in pink, the phosphorous atoms in yellow and green respectively and the lipid chains in grey. The water is not shown for clarity. In panel F, the water molecules within the pore in one of the bilayers are shown in blue. The other pore cannot be seen in the zoom-in but is visible in panel D.</p

Dual Action of BPC194:A Membrane Active Peptide Killing Bacterial Cells

<p>Membrane active peptides can perturb the lipid bilayer in several ways, such as poration and fusion of the target cell membrane, and thereby efficiently kill bacterial cells. We probe here the mechanistic basis of membrane poration and fusion caused by membrane-active, antimicrobial peptides. We show that the cyclic antimicrobial peptide, BPC194, inhibits growth of Gram-negative bacteria and ruptures the outer and inner membrane at the onset of killing, suggesting that not just poration is taking place at the cell envelope. To simplify the system and to better understand the mechanism of action, we performed Forster resonance energy transfer and cryogenic transmission electron microscopy studies in model membranes and show that the BPC194 causes fusion of vesicles. The fusogenic action is accompanied by leakage as probed by dual-color fluorescence burst analysis at a single liposome level. Atomistic molecular dynamics simulations reveal how the peptides are able to simultaneously perturb the membrane towards porated and fused states. We show that the cyclic antimicrobial peptides trigger both fusion and pore formation and that such large membrane perturbations have a similar mechanistic basis.</p>