Evaluation of the Effect of Antibacterial Peptides on Model Monolayers.

The aim of the study was to assess the effect of the synthesized antibacterial peptides: P2 (WKWK)2-KWKWK-NH2, P4 (C12)2-KKKK-NH2, P5 (KWK)2-KWWW-NH2, and P6 (KK)2-KWWW-NH2 on the physicochemical properties of a model biological membrane made of azolectin or lecithin. The Langmuir Wilhelmy method was used for the experiments. Based on the compressibility factor, it was determined that the monolayers formed of azolectin and peptides in the aqueous subphase are in the condensed liquid phase. At the boundary between the condensed and expanded liquid phases, there was a monolayer made of lecithin and P4, P5 or P6 in the aqueous subphase. In turn, the film consisting of lecithin alone (37.7 mN/m) and lecithin and P2 (42.6 mN/m) in the water subphase was in the expanded liquid phase. All peptides change, to varying degrees, the organization and packing of molecules in the monolayer, both those made of azolectin and of lecithin. The test results can be used for further research to design a system with the expected properties for specific organisms.

Effect of Newly Synthesized Structures of Peptides on the Stability of the Monolayers Formed.

The aim of the study was to evaluate the effect of the peptide structure (WKWK)2-KWKWK-NH2, P4 (C12)2-KKKK-NH2, P5 (KWK)2-KWWW-NH2, P6 (KK)2-KWWW-NH2 on their physicochemical properties. The thermogravimetric method (TG/DTG) was used, which made it possible to observe the course of chemical reactions and phase transformations occurring during the heating of solid samples. Based on the DSC curves, the enthalpy of the processes occurring in the peptides was determined. The influence of the chemical structure of this group of compounds on their film-forming properties was determined using the Langmuir-Wilhelmy trough method and was followed by molecular dynamics simulation. Evaluated peptides showed high thermal stability and the first significant mass loss occurred only at about 230 °C and 350 °C. The analysis of the compressibility coefficient of individual peptides indicates that all formed peptide monolayers were in the expanded liquid phase. Their maximum compressibility factor was less than 50.0 mN/m. Its highest value of 42.7 mN/m was achieved in a monolayer made of P4. The results obtained in molecular dynamic simulation indicate that non-polar side chains played an important role in the properties of the P4 monolayer, and the same applies to P5, except that a spherical effect was observed here. A slightly different behavior was observed for the P6 and P2 peptide systems, where the type of amino acids present had an influence. The obtained results indicate that the structure of the peptide affected its physicochemical and layer-forming properties.

Synthetic Antimicrobial Immunomodulatory Peptides: Ongoing Studies and Clinical Trials.

The increasingly widespread antimicrobial resistance forces the search for new antimicrobial substances capable of fighting infection. Antimicrobial peptides (AMPs) and their synthetic analogs form an extensive group of compounds of great structural diversity and multifunctionality, different modes of antimicrobial action, and considerable market potential. Some AMPs, in addition to their proven antibacterial, antifungal, and antiviral activity, also demonstrate anti-inflammatory and immunomodulatory capabilities; these are called innate defense regulator (IDR) peptides. IDR peptides stimulate or inhibit the body's immune system, e.g., by stimulating leukocyte migration to the site of infection, driving macrophage differentiation and activation, providing chemotactic action for neutrophils, degranulation and activation of mast cells, altering chemokine and cytokine production, and even induction of angiogenesis and wound healing. Such multifunctional immunomodulatory peptide molecules are currently being investigated and developed. Exploring and utilizing IDR peptides as an indirect weapon against infectious diseases could represent a completely new strategy to cope with the issue of antimicrobial resistance.

Can Immobilized Artificial Membrane Chromatography Support the Characterization of Antimicrobial Peptide Origin Derivatives?

The emergence and spread of multiple drug-resistant bacteria strains caused the development of new antibiotics to be one of the most important challenges of medicinal chemistry. Despite many efforts, the commercial availability of peptide-based antimicrobials is still limited. The presented study aims to explain that immobilized artificial membrane chromatography can support the characterization of antimicrobial peptides. Consequently, the chromatographic experiments of three groups of related peptide substances: (i) short cationic lipopeptides, (ii) citropin analogs, and (iii) conjugates of ciprofloxacin and levofloxacin, with a cell-penetrating peptide were discussed. In light of the discussion of the mechanisms of action of these compounds, the obtained results were interpreted.

Antimicrobial and Antioxidative Activity of Newly Synthesized Peptides Absorbed into Bacterial Cellulose Carrier against Acne vulgaris.

The ongoing search for effective treatment of Acne vulgaris is concentrated, i.a., on natural peptides with antimicrobial properties. The aim of this work was the development of new amino acid derivatives with potential activity on dermal infections against selected microorganisms, including the facultative anaerobe C. acne. The peptides P1-P6 were synthesized via Fmoc solid phase peptide synthesis using Rink amide AM resin, analyzed by RP-HPLC-MS, FTIR, DPPH radical scavenging activity, and evaluated against C. acne and S. aureus, both deposited and non-deposited in BC. Peptides P1-P6 presented a lack of cytotoxicity, antimicrobial activity, or antioxidative properties correlated with selected structural properties. P2 and P4-P6 sorption in BC resulted in variable data, i.a., confirming the prospective topical application of these peptides in a BC carrier.

The Short Lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 Protect HaCaT Keratinocytes from Bacterial Damage Caused by Staphylococcus aureus Infection in a Co-Culture Model.

The search for new antimicrobial strategies is of major importance since there is a growing resistance of both bacteria and fungi to existing antimicrobials. Lipopeptides are promising and potent antimicrobial compounds. For translation into clinically useful molecules, effectiveness of peptide treatment against human infections must be proved in complex in vitro wound models. The aim of this study was to examine if the synthesized short lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 can protect HaCaT keratinocytes from bacterial damage caused by Staphylococcus aureus infection in a coculture model. After 1 h, 24 h, and 48 h incubation, cellular ATP level and release of the cytotoxicity marker LDH as well as the proinflammatory cytokines interleukin-6 and interleukin-1α were measured. Infection of the keratinocytes resulted in strong bacterial damage of HaCaT cells along with low cellular ATP levels and high release of LDH, IL-6, and IL-1α after 24 h and 48 h. Incubation of the infected human keratinocytes with (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 resulted in protection of the keratinocytes from bacterial damage caused by Staphylococcus aureus infection with ATP, LDH, IL-6, and IL-1α levels comparable to the untreated control. Hence, both synthesized lipopeptides are promising candidates with high therapeutic potential in dermatology for the treatment of topical infections.

Efficacy of newly generated short antimicrobial cationic lipopeptides against methicillin-resistant Staphylococcus aureus (MRSA).

INTRODUCTION: Infection caused by methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) is a serious clinical challenge and research to develop new antimicrobials is imperative. METHODS: This study investigated the in vitro and in vivo efficacy of the short cationic dialkyl lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2. The antibacterial efficacy of (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 was evaluated in representative clinical methicillin-susceptible S. aureus and MRSA strains by both in vitro (MIC, time-kill curve) and in vivo (wax worms model) approaches. RESULTS: These studies revealed that both (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 have rapid bactericidal activity, with a decrease of > 3 log10 colony forming units (CFU)/mL achieved in the first 6 hours of treatment. Furthermore, (C10)2-KKKK-NH2 performed similarly to daptomycin, with a sustained bacterial killing after 24 hours. Wax worms infected and treated with these lipopeptides showed a decreased survival rate of 90% to 50% within the first day of treatment. Scanning electron microscopy determined that the effect of the short lipopeptides in S. aureus was associated with important morphological structural changes that may suggest cell membrane perturbation. CONCLUSION: These findings suggest that the short lipopeptides (C10)2-KKKK-NH2 and (C12)2-KKKK-NH2 may be potential new options for treating MRSA infections.

Are the short cationic lipopeptides bacterial membrane disruptors? Structure-Activity Relationship and molecular dynamic evaluation.

Short cationic lipopeptides are amphiphilic molecules that exhibit antimicrobial activity mainly against Gram-positives. These compounds bind to bacterial membranes and disrupt their integrity. Here we examine the structure-activity relation (SAR) of lysine-based lipopeptides, with a prospect to rationally design more active compounds. The presented study aims to explain how antimicrobial activity of lipopeptides is affected by the charge of lipopeptide headgroup and the length of lipopeptide acyl chain. The obtained SAR models suggest that the lipophilicity of short synthetic cationic lipopeptides is the major factor that determines their antimicrobial activities. In order to link the differences in antimicrobial activity to the mechanism of action of lipopeptides containing one and two hydrophobic chains, we additionally performed molecular dynamic (MD) simulations. By using combined coarse-grained and all-atom simulations we also show that these compounds neither affect the organization of the membrane lipids nor aggregate to form separate phases. These results, along with the onset of antimicrobial activity of lipopeptides well below the critical micelle concentration (CMC), indicate that lipopeptides do not act in a simple detergent-like manner.

Characterization of antimicrobial and hemolytic properties of short synthetic cationic lipopeptides based on QSAR/QSTR approach.

In this study, we investigated the influence of molecular descriptors of cationic lipopeptides on their antimicrobial activity and hemolytic properties. The quantitative structure-activity relationship and quantitative structure-property relationship models were constructed. The antimicrobial, hemolytic and retention data were used as dependent variable and structural parameters as the independent ones. The obtained results suggest that the chromatographic indexes can be employed for prediction of antibacterial activity and that lipopeptides present nonspecific interaction between erythrocytes and bacterial membranes.

Synthesis and Surface Activity of Cationic Amino Acid-Based Surfactants in Aqueous Solution.

I studied the possibility of using amino acid-based surfactants as emulsifiers at the same time as preservatives. Fourteen lipopeptides were synthesized employing a solid phase peptide synthesis procedure. All compounds were designed to be positively charged from +1 to +4 and acylated with fatty acid chain-palmitic and miristic. The surface activity of the obtained lipopeptides was tested using a semi-automatic tensiometer to calculate parameters describing the behavior of lipopeptides in the air/water interface. Such parameters as CMC, surface tension at the CMC point (σCMC), effectiveness (πCMC), and efficiency (pC20) were measured. Emulsifying properties of all lipopeptides were also examined. The studies reveal that the surface active properties of synthesized compounds strongly depend on the length of alkyl chains as well as on the composition of amino acid polar heads. The critical micelle concentration decreases with increasing alkyl chain length of lipopeptides with the same polar head. The effectiveness and efficiency decrease when the number of amino acids in the polar head increases. All lipopeptides established a very weak emulsification power and created unstable water/Miglyol 812 and water/paraffin oil emulsions. Results suggest that lipopeptides cannot be used as emulsifiers; nonetheless, it is possible to use them as auxiliary surfactants with disinfectant properties in combination with more potent emulsifiers.

Cationic Net Charge and Counter Ion Type as Antimicrobial Activity Determinant Factors of Short Lipopeptides.

To get a better insight into the antimicrobial potency of short cationic lipopeptides, 35 new entities were synthesized using solid phase peptide strategy. All newly obtained lipopeptides were designed to be positively charged from +1 to +4. This was achieved by introducing basic amino acid - lysine - into the lipopeptide structure and had a hydrophobic fatty acid chain attached. Lipopeptides were subjected to microbiological tests using reference strains of Gram-negative bacteria: Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Gram-positive bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis, and fungi: Candida albicans, Candida tropicalis, Aspergillus brasiliensis. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimal fungicidal concentration (MFC) were established for each strain. The toxicity toward human cells was determined by hemolysis tests via minimum hemolytic concentration (MHC) determination. The effect of the trifluoroacetic acid (TFA) counter ion on the antimicrobial activity of lipopeptides was also examined by its removing and performing the antimicrobial tests using counter ion-free compounds. The study shows that lipopeptides are more potent against Gram-positive than Gram-negative strains. It was revealed that positive charge equals at least +2 is a necessary condition to observe significant antimicrobial activity, but only when it is balanced with a proper length of hydrophobic fatty acid chain. The hemolytic activity of lipopeptides strongly depends on amino acid composition of the hydrophilic portion of the molecule as well as fatty acid chain length. Compounds endowed with a greater positive charge were more toxic to human erythrocytes. This should be considered during new lipopeptide molecules design. Our studies also revealed the TFA counter ion has no significant effect on the antimicrobial behavior of cationic lipopeptides.

Antimicrobial Peptides Under Clinical Trials.

Today microbial drug resistance has become a serious problem not only within inpatient setting but also within outpatient setting. Repeated intake and unnecessary usage of antibiotics as well as the transfer of resistance genes are the most important factors that make the microorganisms resistant to conventional antibiotics. A large number of antimicrobials successfully used for prophylaxis and therapeutic purposes have now become ineffective [1, 2]. Therefore, new molecules are being studied to be used in the treatment of various diseases. Some of these molecules are structural compounds based on a combination of peptides, for example, naturally occurring endogenous peptide antibiotics and their synthetic analogues or molecules designed de novo using QSAR (quantitative structureproperty relationships)-based methods [3]. Trying to exploit numerous advantages of antimicrobial peptides such as high potency and selectivity, broad range of targets, potentially low toxicity and low accumulation in tissues, pharmaceutical industry aims to develop them as commercially available drugs and appropriate clinical trials are being conducted [4]. In this paper we define clinical trials steps and describe current status of several antimicrobial peptides under clinical development as well as briefly depict peptide drug formulation.

Biological and surface-active properties of double-chain cationic amino acid-based surfactants.

Cationic amino acid-based surfactants were synthesized via solid phase peptide synthesis and terminal acylation of their α and ε positions with saturated fatty acids. Five new lipopeptides, N-α-acyl-N-ε-acyl lysine analogues, were obtained. Minimum inhibitory concentration and minimum bactericidal (fungicidal) concentration were determined on reference strains of bacteria and fungi to evaluate the antimicrobial activity of the lipopeptides. Toxicity to eukaryotic cells was examined via determination of the haemolytic activities. The surface-active properties of these compounds were evaluated by measuring the surface tension and formation of micelles as a function of concentration in aqueous solution. The cationic surfactants demonstrated diverse antibacterial activities dependent on the length of the fatty acid chain. Gram-negative bacteria and fungi showed a higher resistance than Gram-positive bacterial strains. It was found that the haemolytic activities were also chain length-dependent values. The surface-active properties showed a linear correlation between the alkyl chain length and the critical micelle concentration.