Membrane-active Antimicrobial Peptides Research Articles

The Designed Pore-Forming Antimicrobial Peptide C14R Combines Excellent Activity against the Major Opportunistic Human Pathogen Pseudomonas aeruginosa with Low Cytotoxicity.

The diminishing portfolio of mankind's available antibiotics urges science to develop novel potent drugs. Here, we present a peptide fitting the typical blueprint of amphipathic and membrane-active antimicrobial peptides, denominated C14R. This 2 kDa peptide consists of 16 amino acid residues, with seven being either hydrophobic, aromatic, or non-polar, and nine being polar or positively charged, strictly separated on opposite sides of the predicted α-helix. The affinity of the peptide C14R to P. aeruginosa membranes and its intrinsic tendency to productively insert into membranes of such composition were analyzed by dynamic simulations. Its biological impact on the viability of two different P. aeruginosa reference strains was demonstrated by determining the minimal inhibitory concentrations (MICs), which were found to be in the range of 10-15 µg/mL. C14R's pore-forming capability was verified in a permeabilization assay based on the peptide-triggered uptake of fluorescent dyes into the bacterial cells. Finally, the peptide was used in radial diffusion assays, which are commonly used for susceptibility testing of antimicrobial peptides in clinical microbiology. In comparison to reference strains, six clinical P. aeruginosa isolates were clearly affected, thereby paving the way for further in-depth analyses of C14R as a promising new AMP drug in the future.

Design and synthesis of broad-spectrum antimicrobial amphiphilic peptidomimetics to combat drug-resistance

The gradual depletion of antibiotic discovery pipeline makes the antibiotic resistance a difficult clinical problem and a global health emergency. The membrane-active antimicrobial peptides (AMPs) attracted much attention due to a lower tendency to bacterial resistance than traditional antibiotics. However, some immanent drawbacks of AMPs may hamper their application in combating antibiotic resistance in the long run, such as susceptible to enzymatic degradation and low cell permeability. Herein, we report the design and synthesis of a novel series of amphiphilic peptidomimetics, from which we identified compounds that exhibited potent antimicrobial activity against a panel of clinically relevant Gram-positive and Gram-negative bacteria strains. The most potent compound 20 (SD-110-12) is able to kill intracellular bacterial pathogens and prevent the development of bacterial resistance under the tested conditions by targeting cell membranes. Additionally, compound 20 (SD-110-12) obtains good in vivo efficacy that is comparative to vancomycin by eradicating MRSA and suppressing inflammation in a mice infected skin wound model, demonstrating its promising therapeutic potential.

Conjugates of Aminoglycosides with Stapled Peptides as a Way to Target Antibiotic-Resistant Bacteria.

The misuse and overuse of antibiotics led to the development of bacterial resistance to existing aminoglycoside (AMG) antibiotics and limited their use. Consequently, there is a growing need to develop effective antimicrobials against multidrug-resistant bacteria. To target resistant strains, we propose to combine 2-deoxystreptamine AMGs, neomycin (NEO) and amikacin (AMK), with a membrane-active antimicrobial peptide anoplin and its hydrocarbon stapled derivative. The AMG-peptide hybrids were conjugated using the click chemistry reaction in solution to obtain a non-cleavable triazole linker and by disulfide bridge formation on the resin to obtain a linker cleavable in the bacterial cytoplasm. Homo-dimers connected via disulfide bridges between the N-terminus thiol analogues of anoplin and hydrocarbon stapled anoplin were also synthesized. These hybrid compounds show a notable increase in antibacterial and bactericidal activity, as compared to the unconjugated ones or their combinations, against Gram-positive and Gram-negative bacteria, especially for the strains resistant to AMK or NEO. The conjugates and disulfide peptide dimers exhibit low hemolytic activity on sheep red blood erythrocytes.

Peptide Flexibility and the Hydrophobic Moment are Determinants to Evaluate the Clinical Potential of Magainins.

Using a flexibility prediction algorithm and in silico structural modeling, we have calculated the intrinsic flexibility of several magainin derivatives. In the case of magainin-2 (Mag-2) and magainin H2 (MAG-H2) we have found that MAG-2 is more flexible than its hydrophobic analog, Mag-H2. This affects the degree of bending of both peptides, with a kink around two central residues (R10, R11), whereas, in Mag-H2, W10 stiffens the peptide. Moreover, this increases the hydrophobic moment of Mag-H2, which could explain its propensity to form pores in POPC model membranes, which exhibit near-to-zero spontaneous curvatures. Likewise, the protective effect described in DOPC membranes for this peptide regarding its facilitation in pore formation would be related to the propensity of this lipid to form membranes with negative spontaneous curvature. The flexibility of another magainin analog (MSI-78) is even greater than that of Mag-2. This facilitates the peptide to present a kind of hinge around the central F12 as well as a C-terminal end prone to be disordered. Such characteristics are key to understanding the broad-spectrum antimicrobial actions exhibited by this peptide. These data reinforce the hypothesis on the determinant role of spontaneous membrane curvature, intrinsic peptide flexibility, and specific hydrophobic moment in assessing the bioactivity of membrane-active antimicrobial peptides.

Removal and identification of external protein corona members from RBC‐derived extracellular vesicles by surface manipulating antimicrobial peptides

AbstractIn the last years, extracellular vesicles (EVs), secreted by various cells and body fluids have shown extreme potential in biomedical applications. Increasing number of studies suggest that a protein corona could adhere to the surface of EVs which can have a fundamental effect on their function, targeting and therapeutical efficacy. However, removing and identifying these corona members is currently a challenging task to achieve. In this study we have employed red blood cell‐derived extracellular vesicles (REVs) as a model system and three membrane active antimicrobial peptides (AMPs), LL‐37, FK‐16 and CM15, to test whether they can be used to remove protein corona members from the surface of vesicles. These AMPs were reported to preferentially exert their membrane‐related activity via one of the common helical surface‐covering models and do not significantly affect the interior of lipid bilayer bodies. The interaction between the peptides and the REVs was followed by biophysical techniques, such as flow‐linear dichroism spectroscopy which provided the effective applicable peptide concentration for protein removal. REV samples were then subjected to subsequent size exclusion chromatography and to proteomics analysis. Based on the comparison of control REVs with the peptide treated samples, seventeen proteins were identified as external protein corona members. From the three investigated AMPs, FK‐16 can be considered as the best candidate to further optimize EV‐related applicability of AMPs. Our results on the REV model system envisage that membrane active peptides may become a useful set of tools in engineering and modifying surfaces of EVs and other lipid‐based natural particles.

Residual Interactions of LL-37 with POPC and POPE:POPG Bilayer Model Studied by All-Atom Molecular Dynamics Simulation.

LL-37 is a membrane-active antimicrobial peptide (AMP) that could disrupt the integrity of bacterial membranes due to its inherent cationic and amphipathic nature. Developing a shorter derivative of a long peptide such as LL-37 is of great interest, as it can reduce production costs and cytotoxicity. However, more detailed information about the residual interaction between LL-37 and the membrane is required for further optimization. Previously, molecular dynamics simulation using mixed all-atom and united-atom force fields showed that LL-37 could penetrate the bilayer membrane. This study aimed to perform all-atom molecular dynamics simulations, highlighting the residual interaction of LL-37 with the simplest model of the bacterial membrane, POPE:POPG (2:1), and compare its interaction with the POPC, which represents the eukaryotic membrane. The result showed leucine-leucine as the leading residues of LL-37 that first contact the membrane surface. Then, the cationic peptide of LL-37 started to penetrate the membrane by developing salt bridges between positively charged amino acids, Lys-Arg, and the exposed phosphate group of POPE:POPG, which is shielded in POPC. Residues 18 to 29 are suggested as the core region of LL-37, as they actively interact with the POPE:POPG membrane, not POPC. These results could provide a basis for modifying the amino acid sequence of LL-37 and developing a more efficient design for LL-37 derivatives.

Transmembrane peptide effects on bacterial membrane integrity and organization

As the bacterial multidrug resistance crisis continues, membrane-active antimicrobial peptides are being explored as an alternate treatment to conventional antibiotics. In contrast to antimicrobial peptides, which function by a nonspecific membrane disruption mechanism, here we describe a series of transmembrane (TM) peptides that are designed to act as drug efflux inhibitors by aligning with and out-competing a conserved TM4-TM4 homodimerization motif within bacterial small multidrug resistance proteins. The peptides contain two terminal tags: a C-terminal lysine tag to direct the peptides toward the negatively charged bacterial membrane, and an uncharged N-terminal sarcosine (N-methyl-glycine) tag to promote membrane insertion. While effective at inhibiting efflux activity, ostensibly through their designed mechanism of action, the impact of the peptides on the bacterial inner membrane remains undetermined. To evaluate the extant peptide-membrane interactions, we performed a series of biophysical measurements. Circular dichroism spectroscopy and Trp fluorescence showed that the peptides insert into the membrane generally in helical form. Interestingly, differential scanning calorimetry of the peptides added to bacterial-like membranes (POPE:POPG 3:1) revealed the peptides’ ability to demix the POPE and POPG lipids, creating two pools, one of which is likely a peptide-POPG conglomerate, and the other a POPE-rich component where the native POPG content has been depleted. However, dye leakage assays confirmed that these events occur without causing significant membrane disruption both in vitro and in vivo, indicating that the peptides can target the small multidrug resistance TM4-TM4 motif without nonspecific membrane disruption. In related studies, DiOC2(3) fluorescence indicated moderate peptide-mediated reduction of the proton motive force for all peptides, including control peptides that did not display inhibitory activity. The overall findings suggest that peptides designed with suitable tags, sequence hydrophobicity, and charge distribution can be directed more generally to impact proteins whose function involves membrane-embedded protein-protein interactions.

Delineating the Mechanism of Action of a Protease Resistant and Salt Tolerant Synthetic Antimicrobial Peptide against Pseudomonas aeruginosa.

Rapidly growing antimicrobial resistance (AMR) against antibiotics has propelled the development of synthetic antimicrobial peptides (AMPs) as potential antimicrobial agents. An antimicrobial peptide Nle-Dab-Trp-Nle-Dab-Dab-Nle-CONH2 (P36; Nle = norleucine, Dab = diaminobutyric acid, Trp = tryptophan) potent against Pseudomonas aeruginosa (P. aeruginosa) has been developed in the present study. Rational design strategy adopted in this study led to the improvisation of the therapeutic qualities such as activity, salt tolerance, cytotoxicity, and protease resistance of the template peptide P4, which was earlier reported from our group. P36 exhibited salt tolerant antimicrobial potency against P. aeruginosa, along with very low cytotoxicity against mammalian cell lines. P36 was found to be nonhemolytic and resistant toward protease degradation which qualified it as a potent antimicrobial agent. We have investigated the mechanism of action of this molecule in detail using several experimental techniques (spectroscopic, biophysical, and microscopic) and molecular dynamics simulations. P36 was a membrane active AMP with membrane destabilization and deformation abilities, leading to leakage of the intracellular materials and causing eventual cell death. The interaction between P36 and the microbial membrane/membrane mimics was primarily driven by electrostatics. P36 was unstructured in water and upon binding to the microbial membrane mimic SDS, suggesting no influence of secondary structure on its antimicrobial potency. Positive charge, optimum hydrophobic–hydrophilic balance, and chain length remained the most important concerns to be addressed while designing small cationic antimicrobial peptides.

An Efficient Approach for the Design and Synthesis of Antimicrobial Peptide-Peptide Nucleic Acid Conjugates.

Peptide-Peptide Nucleic Acid (PNA) conjugates targeting essential bacterial genes have shown significant potential in developing novel antisense antimicrobials. The majority of efforts in this area are focused on identifying different PNA targets and the selection of peptides to deliver the peptide-PNA conjugates to Gram-negative bacteria. Notably, the selection of a linkage strategy to form peptide-PNA conjugate plays an important role in the effective delivery of PNAs. Recently, a unique Cysteine- 2-Cyanoisonicotinamide (Cys-CINA) click chemistry has been employed for the synthesis of cyclic peptides. Considering the high selectivity of this chemistry, we investigated the efficiency of Cys-CINA conjugation to synthesize novel antimicrobial peptide-PNA conjugates. The PNA targeting acyl carrier protein gene (acpP), when conjugated to the membrane-active antimicrobial peptides (polymyxin), showed improvement in antimicrobial activity against multidrug-resistant Gram-negative Acinetobacter baumannii. Thus, indicating that the Cys-CINA conjugation is an effective strategy to link the antisense oligonucleotides with antimicrobial peptides. Therefore, the Cys-CINA conjugation opens an exciting prospect for antimicrobial drug development.

Membrane-active amino acid-coupled polyetheramine derivatives with high selectivity and broad-spectrum antibacterial activity

Membrane active antimicrobial peptide mimics have been considered as promising alternatives to antibiotics, which interact with bacterial cell membranes to combat bacteria and avoid the emergence of multidrug-resistant bacteria. Herein, a series of star-shaped and membrane-active cationic polyetheramides derived from amino acids, were synthesized via condensation of amino acids and polyetheamine (T403). The antibacterial and anti-biofilm activitives as well as the biocompatibility of these amino acids derived polyetheramides (AAPEAs) were investigated in detail. The star-shaped AAPEAs showed high-efficient and broad-spectrum antibacterial activity against the Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species (ESKAPE) pathogens. In addition, the antibacterial activity was significantly affected by the type of amino acid. L-Trp-T403, which was obtained from L-tryptophan and polyetheramine, exhibited the best antibacterial activity with the minimum inhibitory concentration (MIC) of 1 µg/mL against methicillin-resistant S. aureus (MRSA). Time-kill kinetics and multi-passage resistance tests experiments indicated that L-Trp-T403 could rapidly kill bacteria within 1 h. This compound also showed potent antibacterial activity against bacteria over many passages. Moreover, the AAPEAs exhibited outstanding stability and long-term antibacterial activity in complex mammalian body fluids, as well as good biocompatibility, low hemolytic activity, slight toxicity for mammalian cell (L929) and low in vivo toxicity. The antibacterial activity of L-Trp-T403 was found to be based on the disruption of bacterial membranes, which leads to the leakage of the internal cytoplasm. The AAPEAs possessed high antibacterial and anti-biofilm activity, thus, they are promising to be used as long-term and biofilm-disrupting antimicrobial agents. Statement of significanceThe growing epidemic of MDR-bacteria is becoming a severe public health threat. Here, a series of amino acids derived polyetheramides (AAPEAs) with a star-shaped polyether amide scaffold was synthesized. The star-shaped AAPEAs displayed broad-spectrum antibacterial activity against Gram-positive, Gram-negative bacteria and drug-resistant bacteria MRSA. Notably, the star-shaped AAPEAs were stable under plasma conditions and showed outstanding stability and long-term antibacterial activity in various complex mammalian fluids. Moreover, these star-shaped AAPEAs not only inhibited the formation of biofilms but also disrupted the established biofilms. Furthermore, the membrane-active AAPEAs eradicated bacteria via the fast membrane lytic mechanism, thus plausibly overcoming the MDR effect. These results demonstrate that membrane-active AAPEAs can serve as emerging long-term and biofilm-disrupting antimicrobial agents to treat biofilm-related infections.

In-cell DNP NMR reveals multiple targeting effect of antimicrobial peptide

Dynamic nuclear polarization NMR spectroscopy was used to investigate the effect of the antimicrobial peptide (AMP) maculatin 1.1 on E. coli cells. The enhanced 15N NMR signals from nucleic acids, proteins and lipids identified a number of unanticipated physiological responses to peptide stress, revealing that membrane-active AMPs can have a multi-target impact on E. coli cells. DNP-enhanced 15N-observed 31P-dephased REDOR NMR allowed monitoring how Mac1 induced DNA condensation and prevented intermolecular salt bridges between the main E. coli lipid phosphatidylethanolamine (PE) molecules. The latter was supported by similar results obtained using E. coli PE lipid systems. Overall, the ability to monitor the action of antimicrobial peptides in situ will provide greater insight into their mode of action.

Membrane perturbation, altered morphology and killing of Staphylococcus epidermidis upon contact with a cytocompatible peptide-based antibacterial surface

One possibility to prevent prosthetic infections is to produce biomaterials resistant to bacterial colonization by anchoring membrane active antimicrobial peptides (AMPs) onto the implant surface. In this perspective, a deeper understanding of the mode of action of the immobilized peptides should improve the development of AMP-inspired infection-resistant biomaterials. The aim of the present study was to characterize the bactericidal mechanism against Staphylococcus epidermidis of the AMP BMAP27(1–18), immobilized on titanium disks and on a model resin support, by applying viability counts, Field Emission Scanning Electron Microscopy (FE-SEM), and a fluorescence microplate assay with a membrane potential-sensitive dye. The cytocompatibility to osteoblast-like MG-63 cells was investigated in monoculture and in co-culture with bacteria. The impact of peptide orientation was explored by using N- and C- anchored analogues. On titanium, the ∼50 % drop in bacteria viability and dramatically affected morphology indicate a contact-killing action exerted by the N- and C-immobilized peptides to the same extent. As further shown by the fluorescence assay with the resin-anchored peptides, the bactericidal effect was mediated by rapid membrane perturbation, similar to free peptides. However, at peptide MBC resin equivalents the C-oriented analogue proved more effective with more than 99 % killing and maximum fluorescence increase, compared to half-maximum fluorescence with more than 90 % killing produced by the N-orientation. Confocal microscopy analyses revealed 4–5 times better MG-63 cell adhesion on peptide-functionalized titanium both in monoculture and in co-culture with bacteria, regardless of peptide orientation, thus stimulating further studies on the effects of the immobilized BMAP27(1–18) on osteoblast cells.

A Rapid Fluorescence-Based Microplate Assay to Investigate the Interaction of Membrane Active Antimicrobial Peptides with Whole Gram-Positive Bacteria.

Background: Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Although their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexity. The aim of the present study was to develop a rapid fluorescence-based microplate assay to analyze the membrane effects of AMPs in whole Staphylococcus aureus and Staphylococcus epidermidis. Methods: Bacteria were exposed to bactericidal and sub-inhibitory concentrations of two membrane-active AMPs in the presence of the potential-sensitive dye 3,3′-dipropylthiadicarbocyanine iodide (diSC3(5)) and the DNA staining dye propidium iodide (PI), to simultaneously monitor and possibly distinguish membrane depolarization and membrane permeabilization. Results: The ion channel-forming gramicidin D induced a rapid increase of diSC3(5), but not PI fluorescence, with slower kinetics at descending peptide concentrations, confirming killing due to membrane depolarization. The pore-forming melittin, at sub-MIC and bactericidal concentrations, caused, respectively, an increase of PI fluorescence in one or both dyes simultaneously, suggesting membrane permeabilization as a key event. Conclusions: This assay allowed the distinction between specific membrane effects, and it could be applied in the mode of action studies as well as in the screening of novel membrane-active AMPs.

Antimicrobial Peptides Impair Bacteria Cell Structures within Seconds

Novel antibiotics based on membrane-active antimicrobial peptides (AMPs) are promising candidates for defending the spread of diseases caused by multi-resistant pathogenic bacteria. Notwithstanding the number of works that explore the relationship between AMP activity and membrane architecture, the dynamics and full mechanism that lead to cell death are currently not clear. This prompted us to investigate the cinematographic effects of human lactoferricin derivatives on live E. coli using high-resolution small-angle X-ray and neutron scattering (USAXS/SAXS - VSANS/SANS) measurements and complementary techniques.

Mechanisms of a Small Membrane-Active Antimicrobial Peptide from Hyla punctata

Thousands of antimicrobial peptides have been observed and studied in the past decades; however, their membrane-active mechanisms are ambiguous due to their dynamic structure in the cell membrane. Here, we applied both molecular dynamics (MD) simulations and biophysical experiments to study the small membrane-active antimicrobial peptide Hylaseptin P1 (HSP1), which has significant selectivity towards anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) and bacterial model membranes. HSP1 does not bind and fold onto human red blood cell model membranes, and it only binds, but does not fold, in zwitterionic 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) membranes. This suggests that the lipid chemistry and membrane rigidity are key to prevent HSP1 binding onto membranes, and the lipid headgroup charge may further promote peptide folding in the membrane. Our experiment-validated MD simulations suggest a carpet-like model mechanism for HSP1 through peptide binding, folding, aggregation, and assembly. HSP1 is shorter than the membrane thickness; therefore, the folded peptides aggregate on the surface, cross the membrane, and the oligomeric structure is supported by several surface-bound peptides in both bilayer leaflets.

Curved or linear? Predicting the 3-dimensional structure of α-helical antimicrobial peptides in an amphipathic environment.

α-Helical membrane-active antimicrobial peptides (AMPs) are known to act via a range of mechanisms, including the formation of barrel-stave and toroidal pores and the micellisation of the membrane (carpet mechanism). Different mechanisms imply that the peptides adopt different 3D structures when bound at the water-membrane interface, a highly amphipathic environment. Here, an evolutionary algorithm is used to predict the 3D structure of a range of α-helical membrane-active AMPs at the water-membrane interface by optimising amphipathicity. This amphipathic structure prediction (ASP) is capable of distinguishing between curved and linear peptides solved experimentally, potentially allowing the activity and mechanism of action of different membrane-active AMPs to be predicted. The ASP algorithm is accessible via a web interface at http://atb.uq.edu.au/asp/.

Positive Charge Patterning and Hydrophobicity of Membrane-Active Antimicrobial Peptides as Determinants of Activity, Toxicity, and Pharmacokinetic Stability.

Natural α-helical cationic antimicrobial peptide (CAP) sequences are predominantly amphipathic, with only ca. 2% containing four or more consecutive positively charged amino acids (Lys/Arg). We have designed synthetic CAPs that deviate from these natural sequences, as typified by the charge-clustered peptide KKKKKKAAFAAWAAFAA-NH2, (termed 6K-F17), which displays high antimicrobial activity with no toxicity to mammalian cells. We created a series of peptides varying in charge patterning, increasing the amphipathic character of 6K-F17 to mimic the design of natural CAPs (e.g., KAAKKFAKAWAKAFAA-NH2). Amphipathic sequences displayed increased antimicrobial activity against bacteria but were significantly more toxic to mammalian cells and more susceptible to protease degradation than their corresponding charge-clustered variants, suggesting that amphipathic sequences may be desirable in nature to allow for more versatile functions (i.e., antibacterial, antifungal, antipredator) and rapid clearance from vulnerable host cells. Our approach to clustering of charges may therefore allow for specialization against bacteria, in concert with prolonged peptide half-life.

Correlation of an Antimicrobial Peptide's Potency and Its Influences on Membrane Elasticity

The killing power of membrane-active antimicrobial peptides (AMPs) lies in their ability to disrupt the structures of pathogenic membranes. While understanding the AMPs' mechanisms of action is key to realizing their therapeutic potentials, how the AMPs modulate membrane elasticity and thereby affect membrane structure remains an open question, even though the AMP-induced variations in membrane curvature are widely considered to be crucial. Here, we exploit the x-ray diffraction technique to examine how the dominant bacterial lipid, phosphatidylethanolamine, varies its monolayer elastic properties upon interacting with six artificial peptides mimicking the AMPs' common amino acid content. Remarkably, the monolayer spontaneous curvature ${C}_{0}$ is unaffected by any of the peptides. In contrast, the peptides designated as ${(\mathrm{K}2\mathrm{W})}^{2}$, K4W2, R6, and R9, mimicking the AMP mutants with microbicidal potency, are able to modify the monolayer bending moduli ${K}_{cp}$, while those derived from the impotent mutants cannot. The results are consistent with the scenario that the AMPs disrupt the structure of pathogenic monolayers by, additionally if not exclusively, modulating their ${K}_{cp}$'s, with stiffening and softening leading to two different modes of disruption, that is, toroidal pore formation and membrane micellation, respectively.

Selection and redesign for high selectivity of membrane-active antimicrobial peptides from a dedicated sequence/function database

Antimicrobial peptides (AMPs) are plausible candidates for the development of novel classes of antibiotics with a low tendency to elicit resistance. They often form lesions in the bacterial membrane making it hard for bacteria to develop permanent resistance. However, a potent antibacterial activity is often accompanied by excessive cytotoxicity towards host cells. Modifying known natural sequences, based on desirable biophysical properties, is expensive and time-consuming and often with limited success. ‘Mutator’ is a freely available web-based computational tool for suggesting residue variations that potentially increase a peptide's selectivity, based on the use of quantitative structure activity relationship (QSAR) criteria. Although proven to be successful, it has never been used to analyze multiple sequences simultaneously. Modifying the Mutator algorithm allowed screening of many sequences in the dedicated Database of Anuran Defense Peptides (DADP) and by implementing limited amino acid substitutions on appropriate candidates, propose 8 potentially selective AMPs called Dadapins. Two were chosen for testing, confirming the prediction and validating this approach. They were shown to efficiently inactivate bacteria by disrupting their membranes but to be non-toxic for host cells, as determined by flow cytometry and confirmed by atomic force microscopy (AFM).

Gramicidin S-inspired antimicrobial cyclodextrin to disrupt gram-negative and gram-positive bacterial membranes.

A membrane-active antimicrobial peptide gramicidin S-like amphiphilic structure was prepared from cyclodextrin. The mimic was a cyclic oligomer composed of 6-amino-modified glucose 2,3-di-O-propanoates and it exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria, together with no resistance development and low haemolytic activity against red blood cells.