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The bacterial selectivity of an amphiphilic library of small cyclic α/β-tetra-, α/β-penta-, and α/β-hexapeptides rich in arginine/tryptophan (Arg/Trp) residues, which contains asymmetric backbone configurations and differ in hydrophobicity and alternating d,l-amino acids, was investigated against Bacillus subtilis and Escherichia coli. The structural analyses showed that the peptides tend to form assemblies of different shapes. All-l-peptides, especially the most hydrophobic pentamers, were more strongly anti-B. subtilis. With the exception to cyclo(Phe-d-Trp-β 3 hArg-Arg-d-Trp) (Phe=phenylalanine), the peptides had no effects on inner membrane of E. coli, but lyzed the lipopolysaccharide layer according to their activity pattern. The activities adversely changed with a decrease in the number of amide intramolecular hydrogen bonds in assemblies of diastereomeric peptides and the ratio of hydrophobic/hydrophilic solvent-accessible surface areas. The remarkable enhanced entropic contribution for the partitioning of the least conformationally constrained cyclo(Trp-d-Phe-β 3 hTrp-Arg-d-Arg) sequence into the membranes supported the strong self-assembly behavior, therefore making the peptide less penetrable through the E. coli outer layer., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A great deal of research has been undertaken in order to discover antimicrobial peptides (AMPs) with unexploited mechanisms of action to counteract the health-threatening issues associated with bacterial resistance. The intrinsic effectiveness of AMPs is strongly influenced by their initial interactions with the bacterial cell membrane. Understanding these interactions in the atomistic details is important for the design of the less prone bacteria-resistant peptides. However, these studies always require labor-intensive and difficult steps. With this regard, modeling studies of the AMPs binding to simple lipid membrane systems, e.g., lipid bilayers, is of great advantage. In this chapter, we present an applicable step-by-step protocol to run the molecular dynamics (MD) simulation of the interaction between cyclo-RRWFWR (c-WFW) (a small cyclic AMP) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer using the Groningen machine for chemical simulations (GROMACS) package. The protocol as described here may simply be optimized for other peptide-lipid systems of interest.

The emergence of multiresistant bacteria worldwide together with the shortage of effective antibiotics in the market emphasizes the need for the design and development of the promising agents for the treatment of superbug-associated infections. Antimicrobial peptides (AMPs) have been considered as excellent candidates to tackle this issue, and thousands of peptides of different lengths, amino acid compositions, and mode of action have been discovered and prepared to date. Nevertheless, it is of great importance to develop innovative formulation strategies for delivering these AMPs and to improve their low bioavailability and metabolic stability, particularly against proteases, if these peptides are to find applications in the clinic and administered orally or parenterally or used as dietary supplements. The purpose of this chapter is to describe basic experimental principles, based on analytical reversed-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS), for the prospective design of orally bioavailable AMPs considering the structural characteristics of the peptides and the substrate specificity of proteases that abound in the body especially at sites of infection.

Synthetic bactenecins 1 (HHC-10) and 10 (HHC-36), with excellent activities against bacterial superbugs, display low tryptic stability. To investigate factors influencing this stability, a series of 1/10 derived peptides bearing arginine and lysine analogues with varied methylene chains as well as all-d-isomers were synthesized. Whereas incorporation of d-/l-nonproteinogenic amino acids into the turn-forming peptides did not dramatically affect the antimicrobial activities, the degree of peptide cleavage decreased significantly in peptides with the shortest length of cationic side chain and was influenced by the relative conformational stabilities of the turn structure and the stereoselectivity of tryptic digestion. The site of enzymatic cleavage was located at the less conformationally hindered position distant from the turn motif. Isothermal titration calorimetry showed strong and weak constant increments in the generated heat of enzymatic reaction of unstable and slowly degradable peptides with trypsin, respectively, and suggested a one-site binding model for the enthalpy-driven all-d-peptide-trypsin interactions.

The treatment of infections caused by multi-drugs resistant bacteria and fungi is a particular challenge. Whereas cationic antimicrobial peptides (CAPs) are considered as promising drug candidates for treatment of such superbugs, recent studies have focused on design of those peptides with increased bioavailability and stability against proteases. In between, applications of the quantitative structure-activity relationship (QSAR) studies which provide information on activities of CAPs based on descriptors for each individual amino acid are inevitable. However, the satisfactory results derived from a QSAR model depend highly on the choice of amino acid descriptors and the mathematical strategy used to relate the descriptors to the CAPs' activity. In this study, the quantitative sequence-activity modeling (QSAM) of 60 CAPs derived from O-W-F-I-F-H(1-Bzl)-NH2 sequence which showed excellent activities against a broad range of hazardous microorganisms: e.g., MRSA, MRSE, E. coli and C. albicans, is discussed. The peptides contained natural and non-natural amino acids (AAs) of the both isomers D and L. In this study, a segmented principal component strategy was performed on the structural descriptors of AAs to extract AA's indices. Our results showed that constructed models covered more than 82, 94, 80 and 78 % of the cross-validated variance of C. albicans, MRSA, MRSE and E. coli data sets, respectively. The results were also used to determine the important and significant AAs which are important in CAPs activities. According to the best of our knowledge, it is the first successful attempt in the QSAM studies of peptides containing both natural and non-natural AAs of the both L and D isomers.

Covalent immobilization of cationic antimicrobial peptides (CAPs) at sufficient density and distance from the solid matrix has been suggested as a successful strategy for the generation of biocidal surfaces. To test the hypothesis that the mode of peptide action is decisive for the selection of an appropriate tethering position on solid surfaces, melittin (MEL), a channel-forming peptide, buforin 2 (BUF2), a peptide able to translocate bacterial membranes without permeabilization and targeting nucleic acids, and tritrpticin (TP), described to be membrane-lytic and to have intracellular targets, were C- and N-terminally immobilized on TentaGel S NH(2) resin beads as model surface. The peptide termini were modified with aminooxyacetic acid (AOA) and coupled via oxime-forming ligation. The comparison of the activities of the three peptides and their AOA-modified analogues with a KLAL model peptide which permeabilizes membranes by a so-called "carpet-like" mode provided the following results: The peptides in solution state were active against Bacillus subtilis and Escherichia coli at micromolar concentrations. MEL and TP but not BUF2-derived peptides permeabilized the inner and outer membrane of E. coli and enhanced the permeability of lipid bilayers at concentrations around their antimicrobial values (MICs). Immobilization reduced peptide activity to millimolar MICs. The activity reduction for KLAL was independent of the tethering position and comparably low, as reflected by a low ratio of MIC(tethered)/MIC(free). In contrary, the pore-forming MEL was much less active when immobilized at the N-terminus compared with the C-terminally tethered peptide. C- and N-terminal TP tethering caused an identical but much pronounced activity decrease. The tethered BUF2 peptides were inactive at the tested concentrations suggesting that the peptides could not reach the intracellular targets. In conclusion, membrane active peptides seem to be most suitable for the generation of antimicrobial surfaces, but knowledge about their mode of membrane insertion and positioning is required to identify optimal tethering positions. The relationship between the mechanism of action and position of immobilization is highly relevant for the establishment of a general approach to obtain efficient biocidal solid matrices loaded with CAPs.

Early studies of immobilized peptides mainly focused upon the relationship between structural properties and the activity of soluble and surface-tethered sequences. The intention of this study was to analyze the influence of immobilization parameters upon the activity profile of peptides. Resin beads (TentaGel S NH(2), HypoGel 400 NH(2), and HypoGel 200 NH(2)) with polyethylene glycol spacers of different lengths were rendered antimicrobial by linkage of an amphipathic model KLAL peptide and magainin-derived MK5E. Standard solid-phase peptide synthesis, thioalkylation, and ligation strategies were used to immobilize the peptides at the C and N termini and via different side-chain positions. Depending upon the resin capacity and the coupling strategies, peptide loading ranged between 0.1 and 0.25 micromol/mg for C-terminally and around 0.03 micromol/mg for N-terminally and side-chain-immobilized peptides. Tethering conserved the activity spectra of the soluble peptides at reduced concentrations. The resin-bound peptides were antimicrobial toward Escherichia coli and Bacillus subtilis in the millimolar range compared to the results seen with micromolar concentrations of the free peptides. B. subtilis was more susceptible than E. coli. The antimicrobial activity distinctly decreased with reduction of the spacer length. Slight differences in the antimicrobial effect of KLAL and MK5E bound at different chain positions on TentaGel S NH(2) suggest that the activity is less dependent upon the position of immobilization. Soluble KLAL was active toward red blood cells, whereas MK5E was nonhemolytic at up to about 400 microM. Resin-induced hemolysis hampered the determination of the hemolytic effect of the immobilized peptides. TentaGel S NH(2)-bound peptides enhanced the permeability of the POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline) and mixed POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG) bilayers used to model the charge properties of the biological targets. The results suggest that surface immobilization of the cationic amphipathic antimicrobial peptides does not influence the membrane-permeabilizing mode of action. Peptide insertion into the target membrane and likely the exchange of membrane-stabilizing bivalent cations contribute to the antimicrobial effect. In conclusion, reasonable antimicrobial activity of surface-bound peptides requires the optimization of the coupling parameters, with the length of the spacer and the amount of target-accessible peptide being the most important factors.