Deep learning tools to accelerate antibiotic discovery.

INTRODUCTION: As machine learning (ML) and artificial intelligence (AI) expand to many segments of our society, they are increasingly being used for drug discovery. Recent deep learning models offer an efficient way to explore high-dimensional data and design compounds with desired properties, including those with antibacterial activity. AREAS COVERED: This review covers key frameworks in antibiotic discovery, highlighting physicochemical features and addressing dataset limitations. The deep learning approaches here described include discriminative models such as convolutional neural networks, recurrent neural networks, graph neural networks, and generative models like neural language models, variational autoencoders, generative adversarial networks, normalizing flow, and diffusion models. As the integration of these approaches in drug discovery continues to evolve, this review aims to provide insights into promising prospects and challenges that lie ahead in harnessing such technologies for the development of antibiotics. EXPERT OPINION: Accurate antimicrobial prediction using deep learning faces challenges such as imbalanced data, limited datasets, experimental validation, target strains, and structure. The integration of deep generative models with bioinformatics, molecular dynamics, and data augmentation holds the potential to overcome these challenges, enhance model performance, and utlimately accelerate antimicrobial discovery.

Endocytosis Involved d-Oligopeptide of Tryptophan and Arginine Displays Ordered Nanostructures and Cancer Cell Stereoselective Toxicity by Autophagy.

Owing to their self-aggregation propensity and selective interaction with the anionic membranes, the peptides rich in tryptophan (Trp) and arginine (Arg) are considered for the development of novel anticancer therapeutics. However, the structural insights from the perspective of backbone chirality and spatial orientation of side chains into the selective toxicity of peptides are limited. Here, we investigated the selectivity and cellular uptake of HHC36, a Trp/Arg-rich nonapeptide, and its d-enantiomer (allDHHC36) and a retroinverso analogue in the lung A549 and breast MDA-MB-231 cancer cells. We realized that the d-peptides can specifically induce autophagy at nontoxic concentrations only in the A549 cells supported from the LC 3-II immunostaining expression in the vicinity of the nucleus and the ultrastructural analysis revealing the autophagosome formation. The autophagic flux was also remarkable in the cells exposed to d-peptides at a far lower concentration in synergism with doxorubicin (DOX). In marked contrast, nonselective cell death was observed only if a high amount of HHC36 was applied. HHC36 tended to irregular collagen-like fibrils relative to allDHHC36 that distinctly formed higher-order coiled nanostructures. Interestingly, the short d-peptide fragments were generated in a harsh oxidative condition. Compared with the direct membrane transduction of HHC36, the entry of d-peptides into the lung cancer cells was controlled by endocytosis through the contribution of heparan sulfate proteoglycans (HSPGs) and cholesterol (CHO). However, both l- and d-peptides feasibly crossed the membrane and localized inside the S-phase-arrested cell nucleus. This suggested the likelihood of peptide intercalation with DNA that might differently appear in selective and/or nonselective deaths. These results unraveled the d-handedness-selective toxicity of a self-assembling Trp/Arg-rich sequence that is dependent on the cell type from the aspects of the density of anionic charges and CHO in the outer leaflet of the plasma membrane, as well as the intracellular redox imbalance that may drive the formation of toxic peptide nanostructure fragments.

Turn-folded magainin lipopeptide analog induces cytoplasmic vacuoles in MDA-MB-231 cells through G2-phase arrest.

Selective induced non-canonical programmed deaths in the lipid raft type 1-enriched MDA-MB-231 is a promising treatment approach. Cationic amphiphilic peptides conjugated to relatively long fatty acyl chains that tend to self-aggregate are prone to upregulate necroptotic and paraptotic signaling. We investigated the toxic effects of an N-terminally palmitoylated magainin derivate (P1MK5E) in the MDA-MB-231 cells in relation to its structure at molecular level. The modeling showed that the palmitoylation reinforces a turn-like structural motif in the lipopeptide which is likely required for its activity. P1MK5E triggered intracellular generation of reactive oxygen species (ROS), G2-phase arrest, mitochondrial membrane potential (ΔΨmt) disturbance and presumable flopping of phosphatidylserine (PtdSer) to the cancer cell membrane outer surface in a comparable manner to doxorubicin (DOX) that induces apoptotic signaling. Despite forming extensive congregates of different sizes at the cell surface, P1MK5E had little impacts on the MDA-MB-231 membrane integrity. The cell death upon exposure to the lipopeptide was, however, caspase 3 independent and characterized by cytoplasmic vacuolation and no distinct nuclear fragmentation that is to be privileged in the treatment of apoptotic resistance pathways in triple-negative breast cancers (TNBCs).

CpACpP: In Silico Cell-Penetrating Anticancer Peptide Prediction Using a Novel Bioinformatics Framework.

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).

Palmitoylation of Membrane-Penetrating Magainin Derivatives Reinforces Necroptosis in A549 Cells Dependent on Peptide Conformational Propensities.

Anticancer lipopeptides (ACLPs) are considered promising alternatives to combat resistant cancer cells, but the influence of peptide conformational propensity alone on their selectivity and mechanism remains obscure. In this study, we developed N-palmitoylated MK5E (P1MK5E) and MEK5 (P1MEK5) that have the same composition of 23 residues undergoing the pH-dependent structural alterations but differ in the conformational tendency of their amino acid composites. Nonlipidated peptides were readily accumulated in the A549 cell nucleus by the direct membrane translocation and the heparan sulfate-mediated endocytosis than the lipid-raft-dependent pathway. The increased hydrophobicity favored the amino acid-position-dependent folding of P1MK5E and P1MEK5, respectively, toward the α-helical coiled-coil nanofibrils and amyloidlike ß-protofibrils. At the close concentrations (∼7.5 µM) to the toxic effects of doxorubicin (DOX), P1MK5E exhibited (i) an increased anticancer toxicity through a time-dependent S-phase arrest, (ii) enhanced plasma membrane permeability, and (iii) dose-dependent changes in the cell death characteristic features in the A549 cells relative to P1MEK5 that was almost inactive at ∼75 µM. These observations were in accordance with the TNF-α-mediated necroptotic signaling in the c-MYC/PARP1-overexpressed A549 cells exposed to P1MK5E and accompanied by the ultrastructure of plasma membrane protrusions, extensive endoplasmic reticulum (ER) membrane expansion, mitochondrial swelling, and the formation of distinct cytoplasmic vacuolation. The structural results and the bioactivity behaviors, herein, declared the significance of α-helical propensity in the peptide sequence and the nanostructure morphologies of self-assembling ACLPs upon the selectivity and enhanced anticancer effectiveness, which notably holds promise in the design and development of efficient therapeutics for cancer.

IAMPE: NMR-Assisted Computational Prediction of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are at the focus of attention due to their therapeutic importance and developing computational tools for the identification of efficient antibiotics from the primary structure. Here, we utilized the 13CNMR spectral of amino acids and clustered them into various groups. These clusters were used to build feature vectors for the AMP sequences based on the composition, transition, and distribution of cluster members. These features, along with the physicochemical properties of AMPs were exploited to learn computational models to predict active AMPs solely from their sequences. Naïve Bayes (NB), k-nearest neighbors (KNN), support-vector machine (SVM), random forest (RF), and eXtreme Gradient Boosting (XGBoost) were employed to build the classification system using the collected AMP datasets from the CAMP, LAMP, ADAM, and AntiBP databases. Our results were validated and compared with the CAMP and ADAM prediction systems and indicated that the synergistic combination of the 13CNMR features with the physicochemical descriptors enables the proposed ensemble mechanism to improve the prediction performance of active AMP sequences. Our web-based AMP prediction platform, IAMPE, is available at http://cbb1.ut.ac.ir/.

Bacterial Aggregation Triggered by Fibril Forming Tryptophan-Rich Sequences: Effects of Peptide Side Chain and Membrane Phospholipids.

The influence of side chain residue and phospholipid characteristics of the cytoplasmic membrane upon the fibrillation and bacterial aggregation of arginine (Arg) and tryptophan (Trp) rich antimicrobial peptides (AMPs) has not been well described to date. Here, we utilized the structural advantages of HHC-10 and 4HarHHC-10 (Har, l-homoarginine) that are highly active Trp-rich AMPs and investigated their fibril formation and activity behavior against bacteria. The peptides revealed time-dependent self-assembly of polyproline II (PPII) α-helices, but by comparison, 4HarHHC-10 tended to form higher ordered fibrils due to relatively strong cation-π stacking of Trp with Har residue. Both peptides rapidly killed S. aureus and E. coli at their MICs and caused aggregation of bacteria at higher concentrations. This bacterial aggregation was accompanied by the formation of morphologically distinct electron-dense nanostructures, likely including but not limited to peptides alone. Both HHC-10-derived peptides caused blebs and buds in the E. coli membrane that are rich in POPE phospholipid that promotes negative curvature. However, the main population of S. aureus cells retained their cocci structure upon treatment with HHC peptides even at concentration higher than the MICs. In contrast, the cell aggregation was not induced by HHC fibrils that were most likely stabilized through intra-/intermolecular cation-π stacking. It is proposed that masking of these interactions might have resulted in diminished membrane association/insertion of the HHC nanostructures. The peptides caused aggregation of POPC/POPG (1/3) and POPE/POPG (3/1) liposomes. Nonetheless, disaggregation of the former vesicles was observed at ratios of lipid to peptide of greater than 6 and 24 for HHC-10 and 4HarHHC-10, respectively. Collectively, our results revealed dose-dependent bacterial aggregation mediated by Trp-rich AMPs that was profoundly influenced by the degree of peptide's self-association and the composition and intrinsic curvature of the cytoplasmic membrane lipids.

Arginine/Tryptophan-Rich Cyclic α/ß-Antimicrobial Peptides: The Roles of Hydrogen Bonding and Hydrophobic/Hydrophilic Solvent-Accessible Surface Areas upon Activity and Membrane Selectivity.

The bacterial selectivity of an amphiphilic library of small cyclic α/ß-tetra-, α/ß-penta-, and α/ß-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-ß3 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-ß3 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.

High-Performance Liquid Chromatography and Mass Spectrometry-Based Design of Proteolytically Stable Antimicrobial Peptides.

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.

Molecular Dynamics Simulation and Analysis of the Antimicrobial Peptide-Lipid Bilayer Interactions.

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.

Pronounced peptide selectivity for melanoma through tryptophan end-tagging.

Effects of oligotryptophan end-tagging on the uptake of arginine-rich peptides into melanoma cells was investigated under various conditions and compared to that into non-malignant keratinocytes, fibroblasts, and erythrocytes, also monitoring resulting cell toxicity. In parallel, biophysical studies on peptide binding to, and destabilization of, model lipid membranes provided mechanistic insight into the origin of the selectivity between melanoma and non-malignant cells. Collectively, the results demonstrate that W-tagging represents a powerful way to increase selective peptide internalization in melanoma cells, resulting in toxicity against these, but not against the non-malignant cells. These effects were shown to be due to increased peptide adsorption to the outer membrane in melanoma cells, caused by the presence of anionic lipids such as phosphatidylserine and ganglioside GM1, and to peptide effects on mitochondria membranes and resulting apoptosis. In addition, the possibility of using W-tagged peptides for targeted uptake of nanoparticles/drug carriers in melanoma was demonstrated, as was the possibility to open up the outer membrane of melanoma cells in order to facilitate uptake of low Mw anticancer drugs, here demonstrated for doxorubicin.

Tryptic Stability of Synthetic Bactenecin Derivatives Is Determined by the Side Chain Length of Cationic Residues and the Peptide Conformation.

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.

Quantitative sequence-activity modeling of antimicrobial hexapeptides using a segmented principal component strategy: an approach to describe and predict activities of peptide drugs containing L/D and unnatural residues.

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.

Mode of action of cationic antimicrobial peptides defines the tethering position and the efficacy of biocidal surfaces.

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.

Cyclic antimicrobial R-, W-rich peptides: the role of peptide structure and E. coli outer and inner membranes in activity and the mode of action.

This study compares the effect of cyclic R-, W-rich peptides with variations in amino acid sequences and sizes from 5 to 12 residues upon Gram negative and Gram positive bacteria as well as outer membrane-deficient and LPS mutant Escherichia coli (E. coli) strains to analyze the structural determinants of peptide activity. Cyclo-RRRWFW (c-WFW) was the most active and E. coli-selective sequence and bactericidal at the minimal inhibitory concentration (MIC). Removal of the outer membrane distinctly reduced peptide activity and the complete smooth LPS was required for maximal activity. c-WFW efficiently permeabilised the outer membrane of E. coli and promoted outer membrane substrate transport. Isothermal titration calorimetric studies with lipid A-, rough-LPS (r-LPS)- and smooth-LPS (s-LPS)-doped POPC liposomes demonstrated the decisive role of O-antigen and outer core polysaccharides for peptide binding and partitioning. Peptide activity against the inner E. coli membrane (IM) was very low. Even at a peptide to lipid ratio of 8/1, c-WFW was not able to permeabilise a phosphatidylglycerol/phosphatidylethanolamine (POPG/POPE) bilayer. Low influx of propidium iodide (PI) into bacteria confirmed a low permeabilising ability of c-WFW against PE-rich membranes at the MIC. Whilst the peptide effect upon eukaryotic cells correlated with the amphipathicity and permeabilisation of neutral phosphatidylcholine bilayers, suggesting a membrane disturbing mode of action, membrane permeabilisation does not seem to be the dominating antimicrobial mechanism of c-WFW. Peptide interactions with the LPS sugar moieties certainly modulate the transport across the outer membrane and are the basis of the E. coli selectivity of this type of peptides.

Interaction of W-substituted analogs of cyclo-RRRWFW with bacterial lipopolysaccharides: the role of the aromatic cluster in antimicrobial activity.

The activity of cyclo-RRRWFW (c-WFW) against Escherichia coli has 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 against Bacillus subtilis and erythrocytes increased with increasing hydrophobicity, whereas the effect on E. coli revealed a more complex pattern. Although they had no effect on the E. coli inner 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 of E. 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 against E. coli was 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. coli activity of the cyclic peptides.

Synthesis and thermodynamic characterization of small cyclic antimicrobial arginine and tryptophan-rich peptides with selectivity for Gram-negative bacteria.

One promising strategy to combat the proliferation of bacteria resistance toward current antibiotics is the development of peptide-based drug. Among these compounds is a group of small cyclic peptides rich in arginine (Arg) and tryptophan (Trp) residues with selective toxicity toward Gram-negative bacteria. The small size of these peptides with improved toxicity toward Gram-negative bacteria makes them an interesting candidate to understand the forces responsible for their selectivity and paves the way to develop new therapeutics with potent activity toward multi-resistant Gram-negative bacteria. To reach this goal, isothermal titration calorimetry (ITC) is a useful technique which may provide the complete set of thermodynamic parameters of the interaction of peptides with lipid bilayers mimicking the properties of bacterial membranes within a few hours. The purpose of this chapter is to describe the synthesis of this group of small synthetic antimicrobial peptides together with the application of ITC to study their interaction with lipid membranes.

Immobilization reduces the activity of surface-bound cationic antimicrobial peptides with no influence upon the activity spectrum.

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.