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 NH₂, HypoGel 400 NH₂, and HypoGel 200 NH₂) 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 μmol/mg for C-terminally and around 0.03 μmol/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 towardEscherichia coliandBacillus subtilisin the millimolar range compared to the results seen with micromolar concentrations of the free peptides.B. subtiliswas more susceptible thanE. 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 NH₂suggest 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 μM. Resin-induced hemolysis hampered the determination of the hemolytic effect of the immobilized peptides. TentaGel S NH₂-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.

Cell-penetrating anticancer peptides (Cp-ACPs) are considered promising candidates in solid tumor and hematologic cancer therapies. Current approaches for the design and discovery of Cp-ACPs trust the expensive high-throughput screenings that often give rise to multiple obstacles, including instrumentation adaptation and experimental handling. The application of machine learning (ML) tools developed for peptide activity prediction is importantly of growing interest. In this study, we applied the random forest (RF)-, support vector machine (SVM)-, and eXtreme gradient boosting (XGBoost)-based algorithms to predict the active Cp-ACPs using an experimentally validated data set. The model, CpACpP, was developed on the basis of two independent cell-penetrating peptide (CPP) and anticancer peptide (ACP) subpredictors. Various compositional and physiochemical-based features were combined or selected using the multilayered recursive feature elimination (RFE) method for both data sets. Our results showed that the ACP subclassifiers obtain a mean performance accuracy (ACC) of 0.98 with an area under curve (AUC) ≈ 0.98 vis-à-vis the CPP predictors displaying relevant values of ∼0.94 and ∼0.95 via the hybrid-based features and independent data sets, respectively. Also, the predicting evaluation of Cp-ACPs gave accuracies of ∼0.79 and 0.89 on a series of independent sequences by applying our CPP and ACP classifiers, respectively, which leaves the performance of our predictors better than the earlier reported ACPred, mACPpred, MLCPP, and CPPred-RF. The described consensus-based fusion method additionally reached an AUC of 0.94 for the prediction of Cp-ACP (http://cbb1.ut.ac.ir/CpACpP/Index).

The activity of cyclo-RRRWFW (c-WFW) againstEscherichia colihas been shown to be modulated by the aromatic motif and the lipopolysaccharides (LPS) in the bacterial outer membrane. To identify interaction sites and to elucidate the mode of c-WFW action, peptides were synthesized by the replacement of tryptophan (W) with analogs having altered hydrophobicity, dipole and quadrupole moments, hydrogen-bonding ability, amphipathicity, and ring size. The peptide activity againstBacillus subtilisand erythrocytes increased with increasing hydrophobicity, whereas the effect onE. colirevealed a more complex pattern. Although they had no effect on theE. coliinner membrane even at concentrations higher than the MIC, peptides permeabilized the outer membrane according to their antimicrobial activity pattern, suggesting a major role of LPS in peptide transport across the wall. For isothermal titration calorimetry (ITC) studies of peptide-lipid bilayer interaction, we used POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline), either alone or in mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), to mimic the charge properties of eukaryotic and bacterial membranes, respectively, as well as in mixtures with lipid A, rough LPS, and smooth LPS as models of the outer membrane ofE. coli. Peptide accumulation was determined by both electrostatic and hydrophobic interactions. The susceptibility of the lipid systems followed the order of POPC-smooth LPS >> POPC-rough LPS > POPC-lipid A = POPC-POPG > POPC. Low peptide hydrophobicity and enhanced flexibility reduced binding. The influence of the other properties on the free energy of partitioning was low, but an enhanced hydrogen-bonding ability and dipole moment resulted in remarkable variations in the contribution of enthalpy and entropy. In the presence of rough and smooth LPS, the binding-modulating role of these parameters decreased. The highly differentiated activity pattern againstE. coliwas poorly reflected in peptide binding to LPS-containing membranes. However, stronger partitioning into POPC-smooth LPS than into POPC-rough LPS uncovered a significant role of O-antigen and outer core oligosaccharides in peptide transport and the permeabilization of the outer membrane and the anti-E. coliactivity of the cyclic peptides.