Amram Mor

Improved antimicrobial peptides based on acyl-lysine oligomers

We describe peptidomimetic oligomers that show rapid, nonhemolytic, broad-spectrum bactericidal properties in mice and do not induce the emergence of resistance. The oligomers contain acyl chains, which prevent the formation of stable secondary structure. This design appears advantageous over conventional antimicrobial peptides with respect to in vivo efficacy and safety, and may provide a convenient platform for the development of peptide antibiotics.

Antimicrobial peptide mimics for improved therapeutic properties

The relatively recent recognition of the major role played by antimicrobial peptides (AMPs) in sustaining an effective host response to immune challenges was greatly influenced by studies of amphibian peptides. AMPs are also widely regarded as a potential source of future antibiotics owing to a remarkable set of advantageous properties ranging from molecular simplicity to low-resistance swift-kill of a broad range of microbial cells. However, the peptide formula per se, represents less than ideal drug candidates, namely because of poor bioavailability issues, potential immunogenicity, optional toxicity and high production costs. To address these issues, synthetic peptides have been designed, reproducing the critical peptide biophysical characteristic in unnatural sequence-specific oligomers. Thus, the use of peptidomimetics to overcome the limitations inherent to peptides physical characteristics is becoming an important and promising approach for improving the therapeutic potential of AMPs. Here, we review most recent advances in the design strategies and the biophysical properties of the main classes of mimics to natural AMPs, emphasizing the importance of structure-activity relationship studies in fine-tuning of their physicochemical attributes for improved antimicrobial properties.

Structure-Activity Relationship Study of Antimicrobial Dermaseptin S4 Showing the Consequences of Peptide Oligomerization on Selective Cytotoxicity

To understand how peptide organization in aqueous solution might affect the activity of antimicrobial peptides, the potency of various dermaseptin S4 analogs was assessed against human red blood cells (RBC), protozoa, and several Gram-negative bacteria. Dermaseptin S4 had weak antibacterial activity but potent hemolytic or antiprotozoan effects. K4K20-S4 was 2–3-fold more potent against protozoa and RBC, yet K4K20-S4 was more potent by 2 orders of magnitude against bacteria. K4-S4 had similar behavior as K4K20-S4, but K20-S4 and analogous negative charge substitutions were as active as dermaseptin S4 or had reduced activity. Binding experiments suggested that potency enhancement was not the result of increased affinity to target cells. In contrast, potency correlated well with aggregation properties. Fluorescence studies indicated that K20-S4 and all negative charge substitutions were as aggregated as dermaseptin S4, whereas K4-S4 and K4K20-S4 were clearly less aggregated. Overall, the data indicated that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptides oligomerization in solution and, hence, for its limited spectrum of action. Moreover, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or RBC implied that aggregation can have dramatic consequences on antibacterial activity. Based on these results, we tested the feasibility of selectivity reversal in the activity of dermaseptin S4. Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge affected dramatically the peptides activity and resulted in various analogs that displayed potent antibacterial activity but reduced hemolytic activity. Among these, maximal antibacterial activity was displayed by a 15-mer version that was more potent by 2 orders of magnitude compared with native dermaseptin S4. These results emphasize the notion that peptide-based antibiotics represent a highly modular synthetic antimicrobial system and provide indications of how the peptides physico-chemical properties affect potency and selectivity.

SELECTIVE CYTOTOXICITY OF DERMASEPTIN S3 TOWARD INTRAERYTHROCYTIC PLASMODIUM FALCIPARUM AND THE UNDERLYING MOLECULAR BASIS

The antimicrobial activity of various naturally occurring microbicidal peptides was reported to result from their interaction with microbial membrane. In this study, we investigated the cytotoxicity of the hemolytic peptide dermaseptin S4 (DS4) and the nonhemolytic peptide dermaseptin S3 (DS3) toward human erythrocytes infected by the malaria parasite Plasmodium falciparum. Both DS4 and DS3 inhibited the parasite’s ability to incorporate [3H]hypoxanthine. However, while DS4 was toxic toward both the parasite and the host erythrocyte, DS3 was toxic only toward the intraerythrocytic parasite. To gain insight into the mechanism of this selective cytotoxicity, we labeled the peptides with fluorescent probes and investigated their organization in solution and in membranes. In Plasmodium-infected cells, rhodamine-labeled peptides interacted directly with the intracellular parasite, in contrast to noninfected cells, where the peptides remained bound to the erythrocyte plasma membrane. Binding experiments to phospholipid membranes revealed that DS3 and DS4 had similar binding characteristics. Membrane permeation studies indicated that the peptides were equally potent in permeating phosphatidylserine/phosphatidylcholine vesicles, whereas DS4 was more permeative with phosphatidylcholine vesicles. In aqueous solutions, DS4 was found to be in a higher aggregation state. Nevertheless, both DS3 and DS4 spontaneously dissociated to monomers upon interaction with vesicles, albeit with different kinetics. In light of these results, we propose a mechanism by which dermaseptins permeate cells and affect intraerythrocytic parasites.

Bacterial membranes as predictors of antimicrobial potency.

A wide range of chemical structures having antimicrobial activity have been studied in an effort to treat the increasing emergence of bacteria that are resistant to traditional antibiotics. These agents have varying degrees of toxicity against different bacterial species. We demonstrate, using members of a novel class of antimicrobial agents, the oligomers of acyllysine, that one cause for the difference in species selectivity is the ability to induce the clustering of anionic lipids, resulting in their segregation into domains. This phenomenon occurs only in bacterial membranes composed of both anionic and zwitterionic lipids and not with bacteria whose membrane lipids are largely anionic. As a consequence it can be predicted which bacterial species will be most affected by antimicrobial agents that function principally by this mechanism. This finding allows for the design of new antibiotics with selective toxicity against different groups of bacteria.

Structural Requirements for Potent Versus Selective Cytotoxicity for Antimicrobial Dermaseptin S4 Derivatives

To better understand the structural requirements for selective cytotoxicity of antimicrobial peptides, seven dermaseptin S4 analogs were produced and investigated with respect to molecular organization in solution, binding properties to model phospholipid membranes, and cytotoxic properties. Native dermaseptin S4 displayed high aggregation in solution and high binding affinity. These properties correlated with high cytotoxicity. Yet, potency was progressively limited when facing cells whose plasma membrane was surrounded by increasingly complex barriers. Increasing the positive charge of the native peptide led to partial depolymerization that correlated with higher binding affinity and with virtually non-discriminative high cytotoxicity against all cell types. The C-terminal hydrophobic domain was found responsible for binding to membranes but not for their disruption. Truncations of the C terminus combined with increased positive charge of the N-terminal domain resulted in short peptides having similar binding affinity as the parent compound but displaying selective activity against microbes with reduced toxicity toward human red blood cells. Nuclear magnetic resonance-derived three-dimensional structures of three active derivatives enabled the delineation of a common amphipathic structure with a clear separation of two lobes of positive and negative electrostatic potential surfaces. Whereas the spatial positive electrostatic potential extended considerably beyond the peptide dimensions and was required for potency, selectivity was affected primarily by hydrophobicity. The usefulness of this approach for the design of potent and/or selective cytolytic peptides is discussed herein.

Antibacterial Properties of Dermaseptin S4 Derivatives with In Vivo Activity

ABSTRACT Derivatives of the cytotoxic peptide dermaseptin S4 have recently emerged as potential antimicrobial agents. Here, we report on the antibacterial properties of three derivatives with improved toxicity profiles: a 28-residues K4K20-S4 and two shorter versions, K4-S4(1-16) and K4-S4(1-13). The range of MICs of K4K20-S4 against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were, respectively, 1 to 4, 1 to 4, and 1 to 16 μg/ml. MICs of the short derivatives were rather similar or two to fourfold higher. Each of the three peptides was rapidly bactericidal in vitro, reducing the number of viable CFU of either E. coli or S. aureus by 6 log units in 30 min or less. Compared with MSI-78 or PG-1, K4-S4(1-13) was at least as potent against bacteria (assessed at two MIC multiples) but displayed lesser toxicity against human erythrocytes. Serial passage in subinhibitory concentrations led to emergence of resistance to commercial antibiotics but not to the l- or d isomer of either of the dermaseptin derivatives. The short derivatives were further investigated for antibacterial activity in vivo, using a peritonitis model of mice infected with P. aeruginosa. Naive mice in the vehicle control group exhibited 75% mortality, compared to 18 or 36% mortality in mice that received a single intraperitoneal injection (4.5 mg/kg) of K4-S4(1-16) or K4-S4(1-13), respectively. In vivo bactericidal activity was confirmed in neutropenic mice, where intraperitoneal administration of K4-S4(1-16) reduced the number of viable CFU in a dose-dependent manner by >3 log units within 1 h of exposure, and this was sustained for at least 5 h. Overall, the data suggest that dermaseptin S4 derivatives could be useful in treatment of infections, including infections caused by multidrug-resistant bacteria.

Antimalarial Activities of Dermaseptin S4 Derivatives

ABSTRACT The hemolytic antimicrobial peptide dermaseptin S4 was recently shown to exert antimalarial activity. In this study, we attempted to understand the underlying mechanism(s) and identify derivatives with improved antimalarial activity. A number of dermaseptin S4 derivatives inhibited parasite growth with a 50% inhibitory concentration (IC50) in the micromolar range. Among these, the substituted S4 analog K4K20-S4 was the most potent (IC50 = 0.2 μM), while its shorter version, K4-S4(1–13)a, retained a considerable potency (IC50 = 6 μM). Both K4K20-S4 and K4-S4(1–13)a inhibited growth of the parasites more at the trophozoite stage than at the ring stage. Significant growth inhibition was observed after as little as 1 min of exposure to peptides and proceeded with nearly linear kinetics. The peptides selectively lysed infected red blood cells (RBC) while having a weaker effect on noninfected RBC. Thus, K4K20-S4 lysed trophozoites at concentrations similar to those that inhibited their proliferation, but trophozoites were >30-fold more susceptible than normal RBC to the lytic effect of K4K20-S4, the most hemolytic dermaseptin. The same trend was observed with K4-S4(1–13)a. The d isomers of K4K20-S4 or K4-S4(1–13)a were as active as the l counterparts, indicating that antimalarial activity of these peptides, like their membrane-lytic activity, is not mediated by specific interactions with a chiral center. Moreover, dissipation of transmembrane potential experiments with infected cells indicated that the peptides induce damage in the parasites plasma membrane. Fluorescence confocal microscopy analysis of treated infected cells also indicated that the peptide is able to find its way through the complex series of membranes and interact directly with the intracellular parasite. Overall, the data showed that dermaseptins exert antimalarial activity by lysis of infected cells. Dermaseptin derivatives are also able to disrupt the parasite plasma membrane without harming that of the host RBC.

In Vitro Antiplasmodium Effects of Dermaseptin S4 Derivatives

ABSTRACT The 13-residue dermaseptin S4 derivative K4S4(1-13)a (P) was previously shown to kill intraerythrocytic malaria parasites through the lysis of the host cells. In this study, we have sought peptides that will kill the parasite without lysing the erythrocyte. To produce such peptides, 26 compounds of variable structure and size were attached to the N terminus of P and screened for antiplasmodium and hemolytic activities in cultures of Plasmodium falciparum. Results from this screen indicated that increased hydrophobicity results in amplified antiplasmodium effect, irrespective of the linearity or bulkiness of the additive. However, increased hydrophobicity also was generally associated with increased hemolysis, with the exception of two derivatives: propionyl-P (C3-P) and isobutyryl-P (iC4-P). Both acyl-peptides were more effective than P, with 50% growth inhibition at 3.8, 4.3, and 7.7 μM, respectively. The antiparasitic effect was time dependent and totally irreversible, implying a cytotoxic effect. The peptides were also investigated in parallel for their ability to inhibit parasite growth and to induce hemolysis in infected and uninfected erythrocytes. Whereas the dose dependence of growth inhibition and hemolysis of infected cells overlapped when cells were treated with P, the acyl-peptides exerted 50% growth inhibition at concentrations that did not cause hemolysis. Noticeably, the acyl derivatives, but not P, were able to dissipate the parasite plasma membrane potential and cause depletion of intraparasite potassium under nonhemolytic conditions. These results clearly demonstrate that the acyl-peptides can affect parasite viability in a manner that is dissociated from lysis of the host cell. Overall, the data indicate the potential usefulness of this strategy for development of selective peptides as investigative tools and eventually as antimalarial agents.

Effects of Acyl versus Aminoacyl Conjugation on the Properties of Antimicrobial Peptides

ABSTRACT To investigate the importance of increased hydrophobicity at the amino end of antimicrobial peptides, a dermaseptin derivative was used as a template for a systematic acylation study. Through a gradual increase of the acyl moiety chain length, hydrophobicity was monitored and further modulated by acyl conversion to aminoacyl. The chain lengths of the acyl derivatives correlated with a gradual increase in the peptides global hydrophobicity and stabilization of its helical structure. The effect on cytolytic properties, however, fluctuated for different cells. Whereas acylation gradually enhanced hemolysis of human red blood cells and antiprotozoan activity against Leishmania major, bacteria displayed a more complex behavior. The gram-positive organism Staphylococcus aureus was most sensitive to intermediate acyl chains, while longer acyls gradually led to a total loss of activity. All acyl derivatives were detrimental to activity against Escherichia coli, namely, but not solely, because of peptide aggregation. Although aminoacyl derivatives behaved essentially similarly to the nonaminated acyls, they displayed reduced hydrophobicity, and consequently, the long-chain acyls enhanced activity against all microorganisms (e.g., by up to 12-fold for the aminolauryl derivative) but were significantly less hemolytic than their acyl counterparts. Acylation also enhanced bactericidal kinetics and peptide resistance to plasma proteases. The similarities and differences upon acylation of MSI-78 and LL37 are presented and discussed. Overall, the data suggest an approach that can be used to enhance the potencies of acylated short antimicrobial peptides by preventing hydrophobic interactions that lead to self-assembly in solution and, thus, to inefficacy against cell wall-containing target cells.