Recent advances in antimicrobial peptide-based therapy.

Antimicrobial peptides (AMPs) are a group of polypeptide chains that have the property to target and kill a myriad of microbial organisms including viruses, bacteria, protists, etc. The first discovered AMP was named gramicidin, an extract of aerobic soil bacteria. Further studies discovered that these peptides are present not only in prokaryotes but in eukaryotes as well. They play a vital role in human innate immunity and wound repair. Consequently, they have maintained a high level of intrigue among scientists in the field of immunology, especially so with the rise of antibiotic-resistant pathogens decreasing the reliability of antibiotics in healthcare. While AMPs have promising potential to substitute for common antibiotics, their use as effective replacements is barred by certain limitations. First, they have the potential to be cytotoxic to human cells. Second, they are unstable in the blood due to action by various proteolytic agents and ions that cause their degradation. This review provides an overview of the mechanism of AMPs, their limitations, and developments in recent years that provide techniques to overcome those limitations. We also discuss the advantages and drawbacks of AMPs as a replacement for antibiotics as compared to other alternatives such as synthetically modified bacteriophages, traditional medicine, and probiotics.

The LL-37 Antimicrobial Peptide as a Treatment for Systematic Infection of Acinetobacter baumannii in a Mouse Model.

BACKGROUND: The antimicrobial peptides (AMPs) played a critical role in the innate immunity of the host and are considered natural sources illustrating a broad-spectrum antimicrobial activity with high specificity and low cytotoxicity. AMPs generally possess a net positive charge and have amphipathic structures. Thus, AMPs can bind and interact with negatively charged bacterial cell membranes, leading to destructive defects in biomembranes and ending in cell death. LL37 is the only human cathelicidin-derived antimicrobial peptide that shows a broad spectrum of antimicrobial activity. MATERIALS AND METHODS: To determine the antibacterial efficiency of LL37 in a mouse model of systemic A. baumannii infection, LL37 corresponding gene was expressed in E. coli, purification and refolding situations were optimized. The antimicrobial performance of produced LL-37 against A. baumannii was evaluated in vitro via MIC and Time Kill assays, and its destructive effects on the bacterial cell were confirmed by SEM image. RESULTS: The recombinant LL37 showed strong antibacterial function against A. baumannii at 1.5 µg/mL concentration. Time kill assay showed a sharp reduction of cell viability during the first period of exposure, and complete cell death was recorded after 40 min exposure. CONCLUSION: Furthermore, in vivo results represented a significant ability of LL37 in the treatment of systematic infected mouse models, and all infected mice receiving LL37 protein survived without no trace of bacteria in their blood samples.

Peptides as a therapeutic strategy against Klebsiella pneumoniae.

Increasing levels of resistance to conventional antibiotics have led to a search for new therapeutic options against bacterial infections. Klebsiella pneumoniae is considered a world health problem due to high levels of mortality associated with resistance to multiple antibiotics. Antimicrobial peptides (AMPs) have showed activity against this bacterium, which makes them a promising alternative in tackling resistance. In this article, we carried out an overview of the recent development of AMPs against K. pneumoniae using different designs and acting by different mechanisms, such as a recently proposed one against capsulated strains. Moreover, we outline AMPs' therapeutic potential when tested in combination with conventional antibiotics and against biofilms. Furthermore, challenges and perspectives for applying AMPs in clinical practice are discussed here.

Novel Arginine End-Tagging Antimicrobial Peptides to Combat Multidrug-Resistant Bacteria.

The emergence of multidrug-resistant microorganisms has been termed one of the most common global health threats, emphasizing the discovery of new antibacterial agents. To address this issue, we engineered peptides harboring "RWWWR" as a central motif plus arginine (R) end-tagging and then tested them in vitro and in vivo. Our results demonstrate that Pep 6, one of the engineered peptides, shows great potential in combating Escherichia coli bacteremia and the Staphylococcus aureus skin burn infection model, which induces a 62-90% reduction in bacterial burden. Remarkably, after long serial passages of S. aureus and E. coli for 30 days, Pep 6 is still highly efficient in killing pathogens, compared with 64- and 128-fold increase in minimal inhibitory concentrations (MICs) for vancomycin and polymyxin B, respectively. We also found that Pep 6 exhibited robust biofilm-inhibiting activity and eliminated 61.33% of the mature methicillin-resistant Staphylococcus aureus (MRSA) biofilm with concentration in the MIC level. These results suggest that the RWWWR motif and binding of arginine end-tagging could be harnessed as a new agent for combating multidrug-resistant bacteria.

Evaluation of curcumin and CM11 peptide alone and in combination against amastigote form of Iranian strain of L. major (MRHO/IR75/ER) in vitro.

Curcumin (diferuloylmethane) is the main phytochemical of Curcuma longa Linn, an extract of the rhizome turmeric. For thousands of years, turmeric among other natural products has been used as a dietary spice and as a medicinal plant in Asian countries. The present study reports the leishmanicidal activity of curcumin in different concentrations (10 µM, 20 µM, 40 µM). It is also showing the effect of CM11 peptide (8 µM) alone and in combination with curcumin (10 and 20 µM) as a leishmanicidal drug. The experiments were performed with the amastigote form of Leishmania major (MRHO/IR/75/ER) in vitro and the leishmanicidal activity was analyzed after 12 and 24 h of incubation by Giemsa and DAPI staining. Further investigation was done by using semi-quantitative PCR with new designed common primer pair derived from an 18S rRNA gene belonging to the L. major and mouse, which amplified the above-mentioned gene segments simultaneously with different PCR product size. Our findings showed that curcumin had leishmanicidal activity in a dose and time-dependent manner and its lowest effective dose was at concentrations of 40 µM afetr12 h and 10 µM after 24 h. The IC50 value of curcumin against amastigote forms of L. major was 21.12 µM and 11.77 µM after 12 and 24 h, respectively. Treatment of amastigote form with CM11 (8 µM) alone and in combination with curcumin (10 µM and 20 µM) showed less leishmanicidal activity. Interestingly, CM11 in combination with curcumin (10 µM and 20 µM) had even less leishmanicidal effect compared to curcumin alone in the same concentrations (10 µM and 20 µM). The semi-quantitative PCR analysis confirmed the data achieved by Giemsa and DAPI staining and showed that curcumin reduced the PCR product derived from amastigote form in concentration and time-dependent manner compared to the genome of the host cells.

Agarose oligosaccharide- silver nanoparticle- antimicrobial peptide- composite for wound dressing.

Marine polysaccharides or oligosaccharides have potential to promote wound healing due to their biocompatibility and physicochemical properties. However, microbial infection delays wound healing process, and novel antimicrobial wound dressings are urgently needed. Here, agarose oligosaccharides (AGO) obtained from marine red algae were used as a reducing and stabilizer for green synthesis of silver nanoparticles (AgNPs), and further successfully connected with odorranain A (OA), one of antimicrobial peptides (AMPs), to obtain a novel composite nanomaterial (AGO-AgNPs-OA). Transmission electron microscopy (TEM) and Malvern particle size analyzer showed that AGO-AgNPs-OA was spherical or elliptic with average size of about 100 nm. Circular dichroism (CD) spectroscopy showed that AGO-AgNPs stabilized the α-helical structure of OA. AGO-AgNPs-OA showed stronger anti-bacterial activities than AGO-AgNPs, and had good biocompatibility and significant promoting effect on wound healing. Our data suggest that AMPs conjugated marine oligosaccharides and AgNPs may be effective and safe antibacterial materials for wound therapy.

Antimicrobial Peptides: a New Frontier in Antifungal Therapy.

Invasive fungal infections in humans are generally associated with high mortality, making the choice of antifungal drug crucial for the outcome of the patient. The limited spectrum of antifungals available and the development of drug resistance represent the main concerns for the current antifungal treatments, requiring alternative strategies. Antimicrobial peptides (AMPs), expressed in several organisms and used as first-line defenses against microbial infections, have emerged as potential candidates for developing new antifungal therapies, characterized by negligible host toxicity and low resistance rates. Most of the current literature focuses on peptides with antibacterial activity, but there are fewer studies of their antifungal properties. This review focuses on AMPs with antifungal effects, including their in vitro and in vivo activities, with the biological repercussions on the fungal cells, when known. The classification of the peptides is based on their mode of action: although the majority of AMPs exert their activity through the interaction with membranes, other mechanisms have been identified, including cell wall inhibition and nucleic acid binding. In addition, antifungal compounds with unknown modes of action are also described. The elucidation of such mechanisms can be useful to identify novel drug targets and, possibly, to serve as the templates for the synthesis of new antimicrobial compounds with increased activity and reduced host toxicity.

The Best Peptidomimetic Strategies to Undercover Antibacterial Peptides.

Health-care systems that develop rapidly and efficiently may increase the lifespan of humans. Nevertheless, the older population is more fragile, and is at an increased risk of disease development. A concurrently growing number of surgeries and transplantations have caused antibiotics to be used much more frequently, and for much longer periods of time, which in turn increases microbial resistance. In 1945, Fleming warned against the abuse of antibiotics in his Nobel lecture: "The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant". After 70 years, we are witnessing the fulfilment of Fleming's prophecy, as more than 700,000 people die each year due to drug-resistant diseases. Naturally occurring antimicrobial peptides protect all living matter against bacteria, and now different peptidomimetic strategies to engineer innovative antibiotics are being developed to defend humans against bacterial infections.

Effects of cLFchimera peptide on intestinal morphology, integrity, microbiota, and immune cells in broiler chickens challenged with necrotic enteritis.

Three hundred and sixty 1-day-old male broiler chicks were randomly allocated to 4 treatments of 6 replicates to evaluate the effects of cLFchimera, a recombinant antimicrobial peptide (AMP), on gut health attributes of broiler chickens under necrotic enteritis (NE) challenge. Treatments were as follows: (T1) unchallenged group fed with corn-soybean meal (CSM) without NE challenge and additives (NC); (T2) group fed with CSM and challenged with NE without any additives (PC); (T3) PC group supplemented with 20 mg cLFchimera/kg diet (AMP); (T4) PC group supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet (antibiotic). Birds were sampled for villi morphology, ileal microbiota, and jejunal gene expression of cytokines, tight junctions proteins, and mucin. Results showed that AMP ameliorated NE-related intestinal lesions, reduced mortality, and rehabilitated jejunal villi morphology in NE challenged birds. While the antibiotic non-selectively reduced the count of bacteria, AMP restored microflora balance in the ileum of challenged birds. cLFchimera regulated the expression of cytokines, junctional proteins, and mucin transcripts in the jejunum of NE challenged birds. In conclusion, cLFchimera can be a reliable candidate to substitute growth promoter antibiotics, while more research is required to unveil the exact mode of action of this synthetic peptide.

Cyclic Derivative of Host-Defense Peptide IDR-1018 Improves Proteolytic Stability, Suppresses Inflammation, and Enhances In Vivo Activity.

Host-defense peptides have drawn significant attention as new drugs or drug adjuvants to combat multidrug-resistant bacteria. In this study, we report the development of cyclic derivatives of the immunomodulatory and antibiofilm innate defense regulator peptide (IDR)-1018 based on three different synthetic strategies including head-to-tail cyclization (C1), side-chain-to-tail cyclization (C2), and a disulfide bond cross-linkage (C3). The generated mimetics showed enhanced proteolytic stability and reduced aggregation in vitro and in vivo. The C2 derivative exhibited exceptional ability to suppress inflammation and significantly reduce bacterial loads in a high-density Staphylococcus aureus murine skin infection model. The findings describe different routes to the creation of enzymatically stable mimetics of IDR-1018 and identify a promising new cyclic analogue against bacterial infections.

Enhanced therapeutic index of an antimicrobial peptide in mice by increasing safety and activity against multidrug-resistant bacteria.

The rising prevalence of antibiotic resistance underscores the urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs) are potentially effective therapeutics that disrupt bacterial membranes regardless of resistance to traditional antibiotics. We have developed engineered cationic AMPs (eCAPs) with broad activity against multidrug-resistant (MDR) bacteria, but stability remains an important concern. Therefore, we sought to enhance the clinical utility of eCAP WLBU2 in biological matrices relevant to respiratory infection. A designed substitution of d-Val for l-Val resulted in increased resistance to protease enzymatic degradation. We observed multiple gains of functions such as higher activity against bacteria in biofilm mode of growth, significantly lower toxicity to erythrocytes and white blood cells compared to WLBU2, with increased safety in mice. Direct airway delivery revealed a therapeutic index of >140 for the selected enantiomer compared to that of <35 for WLBU2. The data warrant clinical exploration by aerosolized delivery to mitigate MDR-related respiratory infection.

Romo1-Derived Antimicrobial Peptide Is a New Antimicrobial Agent against Multidrug-Resistant Bacteria in a Murine Model of Sepsis.

To overcome increasing bacterial resistance to conventional antibiotics, many antimicrobial peptides (AMPs) derived from host defense proteins have been developed. However, there are considerable obstacles to their application to systemic infections because of their low bioavailability. In the present study, we developed an AMP derived from Romo1 (AMPR-11) that exhibits a broad spectrum of antimicrobial activity. AMPR-11 showed remarkable efficacy against sepsis-causing bacteria, including multidrug-resistant strains, with low toxicity in a murine model of sepsis after intravenous administration. It seems that AMPR-11 disrupts bacterial membranes by interacting with cardiolipin and lipid A. From the results of this study, we suggest that AMPR-11 is a new class of agent for overcoming low efficacy in the intravenous application of AMPs and is a promising candidate to overcome multidrug resistance.IMPORTANCE Abuse of antibiotics often leads to increase of multidrug-resistant (MDR) bacteria, which threatens the life of human beings. To overcome threat of antibiotic resistance, scientists are developing a novel class of antibiotics, antimicrobial peptides, that can eradicate MDR bacteria. Unfortunately, these antibiotics have mainly been developed to cure bacterial skin infections rather than others, such as life-threatening sepsis. Major pharmaceutical companies have tried to develop antiseptic drugs; however, they have not been successful. Here, we report that AMPR-11, the antimicrobial peptide (AMP) derived from mitochondrial nonselective channel Romo1, has antimicrobial activity against Gram-positive and Gram-negative bacteria comprising many clinically isolated MDR strains. Moreover, AMPR-11 increased the survival rate in a murine model of sepsis caused by MDR bacteria. We propose that AMPR-11 could be a novel antiseptic drug candidate with a broad antimicrobial spectrum to overcome MDR bacterial infection.

Identification of new dermaseptins with self-assembly tendency: membrane disruption, biofilm eradication, and infected wound healing efficacy.

Severe infections associated with antibiotic-resistant bacteria and biofilms have attracted increasing interest as these diseases are difficult to treat with current antibiotics. Typical cationic antimicrobial peptides dermaseptins are considered to be the most promising next-generation antibiotics because of their broad-spectrum antimicrobial activities and minor side effects. Two new dermaseptin peptides, DMS-PS1 and DMS-PS2, have been identified by "shotgun" molecular cloning of encoding cDNAs in the crude skin secretions of the waxy monkey tree frog, Phyllomedusa sauvagei. The mature peptide sequences predicted from the cloned cDNAs were separated from crude skin secretions and confirmed by mass spectrometry. Chemically synthetic replicates were assessed for various biological activities. Both dermaseptins were potently effective against a broad spectrum of microorganisms including antibiotic-resistant bacteria and displayed significant potency against gram-positive and gram-negative bacterial biofilms with low toxicity towards mammalian red blood cells. Remarkably, DMS-PS2 was effective against infections in murine skin caused by methicillin-resistant Staphylococcus aureus as a result of an induced wound. The actions of DMS-PS2 were with a membrane permeabilization mode. Overall, the data provided convincing evidence for the development of anti-infectious agents and/or biomaterials as a new therapeutic approach against bacterial infections. STATEMENT OF SIGNIFICANCE: Bacterial adhesion to biomaterials remains a major problem. Antimicrobial peptides (AMPs) are well-known components of the innate immune system that can be applied to overcome biofilm-associated infections. Cationic dermaseptin peptides showed significant broad-spectrum antimicrobial activities and activities against bacterial biofilms of persistent infections in association with weak toxicity for mammalian red blood cells. The membrane permeabilizing ability of DMS-PS2 was confirmed, and importantly, it demonstrated potent efficiency of the treatment of MRSA infected murine skin model. Furthermore, beyond our expectation, DMS-PS2 showed a self-aggregating parameter, indicating a promising potential for the use of immobilized AMPs in clinical applications., which makes it also a promising suggestion for infection-proof biomaterial development.

Selection of antimicrobial frog peptides and temporin-1DRa analogues for treatment of bacterial infections based on their cytotoxicity and differential activity against pathogens.

Cationic, amphipathic, α-helical host-defense peptides (HDPs) that are naturally secreted by certain species of frogs (Anura) possess potent broad-spectrum antimicrobial activity and show therapeutic potential as alternatives to treat infections by multidrug-resistant pathogens. Fourteen amphibian skin peptides and twelve analogues of temporin-1DRa were studied for their antimicrobial activities against clinically relevant human or animal skin infection-associated pathogens. For comparison, antimicrobial potencies of frog skin peptides against a range of probiotic lactobacilli were determined. We used the VITEK 2 system to define a profile of antibiotic susceptibility for the bacterial panel. The minimal inhibitory concentration (MIC) values of the naturally occurring temporin-1DRa, CPF-AM1, alyteserin-1c, hymenochirin-2B, and hymenochirin-4B for pathogenic bacteria were threefold to ninefold lower than the values for the tested probiotic strains. Similarly, temporin-1DRa and its [Lys4 ], [Lys5 ], and [Aib8 ] analogues showed fivefold to 6.5-fold greater potency against the pathogens. In the case of PGLa-AM1, XT-7, temporin-1DRa and its [D-Lys8 ] and [Aib13 ] analogues, no apoptosis or necrosis was detected in human peripheral blood mononuclear cells at concentrations below or above the MIC. Given the differential activity against commensal bacteria and pathogens, some of these peptides are promising candidates for further development into therapeutics for topical treatment of skin infections.

Antimicrobial peptide, cLF36, affects performance and intestinal morphology, microflora, junctional proteins, and immune cells in broilers challenged with E. coli.

This study investigated the effects of an antimicrobial peptide (AMP), cLF36, on growth performance and the histophysiological changes of the intestine in E. coli-challenged broiler chickens. A total number of 360 day old male chicks were randomly assigned to 4 groups of 6 replicates as follows: T1) negative control diet based on corn-soybean meal without E. coli challenge and additives; T2) positive control diet based on corn-soybean meal and challenged with E. coli without any additives; T3) positive control diet challenged with E. coli and supplemented with 20 mg AMP (cLF36)/kg diet; T4) positive control diet challenged with E. coli and supplemented with 45 mg antibiotic (bacitracin methylene disalicylate)/kg diet. Results showed that T3 improved growth performance and the jejunal morphology of E. coli-challenged chickens similar to those of T4. While antibiotic non-selectively decreased the population of ileal bacteria, AMP increased the population of Lactobacillus spp. and decreased harmful bacteria in the ileum of E. coli-challenged chickens. Supplementing E. coli-challenged chickens with AMP improved the gene expression of immune cells and upregulated the expression of tight junction proteins compared to other challenged groups. In conclusion, although cLF36 beneficially affected growth performance and the intestinal morphology of E. coli-challenged chickens similar to those of the antibiotic group, this AMP drastically improved the intestinal microbiome, immune cells, and junctional proteins compared to other E. coli-challenged birds, and can be nominated as an alternative for growth promoter antibiotics.

Antimicrobial and Antibiofilm Activities of Helical Antimicrobial Peptide Sequences Incorporating Metal-Binding Motifs.

Antimicrobial peptides (AMPs) represent alternative strategies to combat the global health problem of antibiotic resistance. However, naturally occurring AMPs are generally not sufficiently active for use as antibiotics. Optimized synthetic versions incorporating additional design principles are needed. Here, we engineered amino-terminal Cu(II) and Ni(II) (ATCUN) binding motifs, which can enhance biological function, into the native sequence of two AMPs, CM15 and citropin1.1. The incorporation of metal-binding motifs modulated the antimicrobial activity of synthetic peptides against a panel of carbapenem-resistant enterococci (CRE) bacteria, including carbapenem-resistant Klebsiella pneumoniae (KpC+) and Escherichia coli (KpC+). Activity modulation depended on the type of ATCUN variant utilized. Membrane permeability assays revealed that the in silico selected lead template, CM15, and its ATCUN analogs increased bacterial cell death. Mass spectrometry, circular dichroism, and molecular dynamics simulations indicated that coordinating ATCUN derivatives with Cu(II) ions did not increase the helical tendencies of the AMPs. CM15 ATCUN variants, when combined with Meropenem, streptomycin, or chloramphenicol, showed synergistic effects against E. coli (KpC+ 1812446) biofilms. Motif addition also reduced the hemolytic activity of the wild-type AMP and improved the survival rate of mice in a systemic infection model. The dependence of these bioactivities on the particular amino acids of the ATCUN motif highlights the possible use of size, charge, and hydrophobicity to fine-tune AMP biological function. Our data indicate that incorporating metal-binding motifs into peptide sequences leads to synthetic variants with modified biological properties. These principles may be applied to augment the activities of other peptide sequences.

Synthesized natural peptides from amphibian skin secretions increase the efficacy of a therapeutic vaccine by recruiting more T cells to the tumour site.

BACKGROUND: Therapeutic vaccines against cervical cancer remain ineffective. Previously, we demonstrated that blocking the signalling of a cytokine, interleukin 10, at the time of immunisation elicited significantly higher numbers of antigen specific T cells and inhibited tumour growth in mice. RESULTS: In the current paper, we demonstrate, in a HPV16 E6/E7 transformed TC-1 tumour mouse model, that despite increased antigen specific T cell numbers, blocking IL-10 signalling at the time of immunisation does not increase the survival time of the TC-1 tumour bearing mice compared to mice receiving the same immunisation with no IL-10 signalling blockade. Moreover, the function of tumour infiltrating T cells isolated 3 weeks post TC-1 transplantation is more suppressed than those isolated 2 weeks after tumour inoculation. We demonstrate that synthesized caerin peptides, derived from amphibian skin secretions, 1) were able to inhibit TC-1 tumour growth both in vitro and in vivo; 2) are environmentally stable; and 3) promote the secretion of pro-inflammatory interlukine-6 by TC-1 cells. Notably caerin peptides were able to increase the survival time of TC-1 tumour bearing mice after therapeutic vaccination with a HPV16E7 peptide-based vaccine containing IL-10 inhibitor, via recruiting increased levels of T cells to the tumour site. CONCLUSION: Caerin peptides increase the efficacy of a therapeutic vaccine by recruiting more T cells to the tumour site.

Prevention of urinary catheter-associated infections by coating antimicrobial peptides from crowberry endophytes.

Urinary catheters are extensively used in hospitals, being responsible for about 75% of hospital-acquired infections. In this work, a de novo designed antimicrobial peptide (AMP) Chain201D was studied in the context of urinary catheter-associated infections. Chain201D showed excellent antimicrobial activity against relevant ATCC strains and clinical isolates of bacteria and yeast and demonstrated high stability in a wide range of temperatures, pH and salt concentrations. Moreover, the bactericidal activity of Chain201D immobilized on a model surface was studied against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), some of the most prevalent strains found in urinary catheter-associated infections. Chain201D was successfully tethered to ((1-mercapto-11-undecyl)-(tetra(ethylene glycol) (EG4)) terminated self-assembled monolayers (SAMs), (EG4-SAMs), activated by 1,1'-Carbonyldiimidazole (CDI) at different concentrations. Chain201D surfaces can bind and kill by contact a high percentage of adherent bacteria. These achievements are obtained without any peptide modification (for chemoselective conjugation) and without the use of a spacer. Moreover, increased amounts of immobilized AMP lead to higher numbers of adhered/dead bacteria, revealing a concentration-dependent behaviour and demonstrating that Chain201D has excellent potential for developing antimicrobial urinary catheters.

Characterization of the in vitro, ex vivo, and in vivo Efficacy of the Antimicrobial Peptide DPK-060 Used for Topical Treatment.

Antimicrobial peptides, also known as host defense peptides, have recently emerged as a promising new category of therapeutic agents for the treatment of infectious diseases. This study evaluated the preclinical in vitro, ex vivo, and in vivo antimicrobial activity, as well as the potential to cause skin irritation, of human kininogen-derived antimicrobial peptide DPK-060 in different formulations designed for topical delivery. We found that DPK-060 formulated in acetate buffer or poloxamer gel caused a marked reduction of bacterial counts of Staphylococcus aureus in vitro (minimum microbicidal concentration <5 µg/ml). We also found that DPK-060 in poloxamer gel significantly suppressed microbial survival in an ex vivo wound infection model using pig skin and in an in vivo mouse model of surgical site infection (≥99 or ≥94% reduction in bacterial counts was achieved with 1% DPK-060 at 4 h post-treatment, respectively). Encapsulation of DPK-060 in different types of lipid nanocapsules or cubosomes did not improve the bactericidal potential of the peptide under the applied test conditions. No reduction in cell viability was observed in response to administration of DPK-060 in any of the formulations tested. In conclusion, the present study confirms that DPK-060 has the potential to be an effective and safe drug candidate for the topical treatment of microbial infections; however, adsorption of the peptide to nanocarriers failed to show any additional benefits.

Hybrids made from antimicrobial peptides with different mechanisms of action show enhanced membrane permeabilization.

Combining two known antimicrobial peptides (AMPs) into a hybrid peptide is one promising avenue in the design of agents with increased antibacterial activity. However, very few previous studies have considered the effect of creating a hybrid from one AMP that permeabilizes membranes and another AMP that acts intracellularly after translocating across the membrane. Moreover, very few studies have systematically evaluated the order of parent peptides or the presence of linkers in the design of hybrid AMPs. Here, we use a combination of antibacterial measurements, cellular assays and semi-quantitative confocal microscopy to characterize the activity and mechanism for a library of sixteen hybrid peptides. These hybrids consist of permutations of two primarily membrane translocating peptides, buforin II and DesHDAP1, and two primarily membrane permeabilizing peptides, magainin 2 and parasin. For all hybrids, the permeabilizing peptide appeared to dominate the mechanism, with hybrids primarily killing bacteria through membrane permeabilization. We also observed increased hybrid activity when the permeabilizing parent peptide was placed at the N-terminus. Activity data also highlighted the potential value of considering AMP cocktails in addition to hybrid peptides. Together, these observations will guide future design efforts aiming to design more active hybrid AMPs.