Short Cationic Antimicrobial Peptides Research Articles

Cationicity Enhancement on the Hydrophilic Face of Ctriporin Significantly Reduces Its Hemolytic Activity and Improves the Antimicrobial Activity against Antibiotic-Resistant ESKAPE Pathogens.

The ESKAPE pathogen-associated antimicrobial resistance is a global public health issue, and novel therapeutic strategies are urgently needed. The short cationic antimicrobial peptide (AMP) family represents an important subfamily of scorpion-derived AMPs, but high hemolysis and poor antimicrobial activity hinder their therapeutic application. Here, we recomposed the hydrophilic face of Ctriporin through lysine substitution. We observed non-linear correlations between the physiochemical properties of the peptides and their activities, and significant deviations regarding the changes of antimicrobial activities against different bacterial species, as well as hemolytic activity. Most importantly, we obtained two Ctriporin analogs, CM5 and CM6, these two have significantly reduced hemolytic activity and more potent antimicrobial activities against all tested antibiotic-resistant ESKAPE pathogens. Fluorescence experiments indicated they may perform the bactericidal function through a membrane-lytic action model. Our work sheds light on the potential of CM5 and CM6 in developing novel antimicrobials and gives clues for optimizing peptides from the short cationic AMP family.

A Short Cationic Peptide Derived from Cecropin and Melittin Peptides Induce Apoptosis in Jurkat and Raji Leukemia Cell Lines.

The creation of brand-new, potent, and less harmful medications to treat leukemia is urgently needed. Antimicrobial peptides (AMPs) have drawn a lot of interest as potential substitutes for chemotherapy. In the present investigation, the anticancer activity of CM11, a short cationic AMP, was assessed on Jurkat and Raji leukemia cell lines and peripheral blood mononuclear cells (PBMCs). Different CM11 doses were applied to the Jurkat and Raji cell lines and PBMCs throughout a 24-hour period. The impact of the CM11 on cell viability and toxicity was assessed using an MTT assay. Flow cytometry and Real-Time PCR were used to analyze the effect of this peptide on apoptotic/necrosis pathways and assess the ratio expression of the P53 and Bcl-2 genes, respectively. Despite the fact that peptide toxicity was successful in a variety of cell lines, cancer cells were more sensitive to the medication. The survival of Jurkat and Raji cell lines treated with 32 μg/ml peptide was 47% and 51%, respectively, while the survival of normal PBMC cells was about 65%. According to flow cytometry, Jurkat and Raji cells exposed to peptide had much greater levels of apoptosis than PBMCs. Peptide-treated cells were associated with increased expression of P53 the gene and decreased expression of the Bcl-2 gene. These results revealed that the CM11 caused more cytotoxicity to leukemia Raji and Jurkat leukemia cells compared to the normal cells by apoptosis pathway. Our findings demonstrated the potential of CM11 peptide to develop as a new antileukemic agent.

Effect of number of lysine motifs on the bactericidal and hemolytic activity of short cationic antimicrobial peptides.

Antimicrobial peptides (AMPs) are vital components of the nonspecific immune system that represent a promising broad-spectrum alternative to conventional antibiotics. Several short cationic antimicrobial peptides show highly effective antibacterial activity and low hemolytic activity, which are based on the action of a few critical amino acids, such as phenylalanine (F) and lysine (K). Previous studies have reported that Fmoc-based phenylalanine peptides possess appreciable antibacterial potency against Gram-positive bacteria, but their ability to kill Gram-negative bacteria was suboptimal. In this study, we designed and prepared a series of Fmoc-KnF peptide (n=1-3) series by adding lysine motifs to strengthen their broad-spectrum antibacterial activity. The effect was investigated that the amount of lysine in Fmoc-F peptides on their antibacterial properties and hemolytic activities. Our results showed that the Fmoc-KKF peptide holds the strongest antimicrobial activity against both Gram-positive and negative bacteria among all designed peptides, as well as low hemolytic activity. These results provide support for the general strategy of enhancing the broad-spectrum antibacterial activity of AMPs through increased lysine content.

A short novel antimicrobial peptide BP100-W with antimicrobial, antibiofilm and anti-inflammatory activities designed by replacement with tryptophan

BP100 is a short cationic antimicrobial peptide (AMP) designed using a combinatorial chemistry approach based on the cecropin A-melittin hybrid. It displays potent antimicrobial activity against gram-negative bacteria and low toxicity toward eukaryotic cells. To develop a short AMP with potent cell selectivity, antibiofilm and anti-inflammatory activities, we designed a newly BP100 analog, BP100-W, in which Leu-3 at the hydrophobic face of BP100 was replaced by Trp. BP100-W possessed better cell selectivity, with a 1.7-fold higher therapeutic index than BP100. BP100-W displayed more effective synergistic activity when combined with several antibiotics, such as chloramphenicol, ciprofloxacin and oxacillin, compared to BP-100. BP100-W also exhibited stronger antibiofilm activity than BP100 in inhibiting biofilm formation by multidrug-resistant Pseudomonas aeruginosa (MDRPA) and eradicating the preformed biofilms of MDRPA. Moreover, unlike BP100, BP100-W significantly suppressed the production and expression of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines, such as the tumor necrosis factor-α and nitric oxide. Boron-dipyrromethene-TR-cadaverine displacement assay demonstrated that the inhibitory activity of BP100-W on LPS-induced inflammation in RAW 264.7 cells may be due to increased direct interaction with LPS. Our results suggest that BP100-W exhibits potential for future use as an antimicrobial, antibiofilm and anti-inflammatory agent.

Effects of Synthetic Short Cationic Antimicrobial Peptides on the Catalytic Activity of Myeloperoxidase, Reducing Its Oxidative Capacity.

Cationic antimicrobial peptides (CAMPs) have gained attention as promising antimicrobial therapeutics causing lower or no bacterial resistance. Considerable achievements have been made in designing new CAMPs that are highly active as antimicrobials. However, there is a lack of research on their interaction with biologically important proteins. This study focused on CAMPs' effects on myeloperoxidase (MPO), an enzyme which is microbicidal and concomitantly damaging to host biomolecules and cells due to its ability to produce reactive oxygen and halogen species (ROS/RHS). Four CAMPs designed by us were employed. MPO catalytic activity was assessed by an absorbance spectra analysis and by measuring enzymatic activity using Amplex Red- and Celestine Blue B-based assays. The peptide Hm-AMP2 accelerated MPO turnover. Pept_1545 and Hm-AMP8 inhibited both the MPO chlorinating and peroxidase activities, with components of different inhibition types. Hm-AMP8 was a stronger inhibitor. Its Ki towards H2O2 and Cl- was 0.3-0.4 μM vs. 11-20 μM for pept_1545. Peptide tyrosine and cysteine residues were involved in the mechanisms of the observed effects. The results propose a possible dual role of CAMPs as both antimicrobial agents and agents that downregulate MPO activation, and suggest CAMPs as prototypes for the development of antioxidant compounds to prevent MPO-mediated ROS/RHS overproduction.

Influence of adding terminal tags on the structural and antimicrobial properties of the peptide caerin 1.1

Aquaculture is threatened by severe bacterial and viral outbreaks which can cause massive mortality and limit productivity. Antimicrobial peptides are a promising tool for the control of infectious diseases; however, their use in the aquaculture sector has been poorly explored. Recombinant production of peptides is an interesting alternative to their chemical synthesis, which has still important challenges to address. Although extra amino acid tags are usually added to the ends of recombinant proteins to enhance their stability and facilitate their purification or their translational processing, the effect of these tags on the function and the stability of antimicrobial peptides is completely unknown. This is particularly important in the case of the short cationic antimicrobial peptide caerin, originally isolated from frog skin secretions, and other peptides which bioactivity has been directly related with its ability to adopt an amphipathic alpha-helical structure upon interaction with the microbial membranes. Caerin has demonstrated bactericidal activity against several important human pathogens, however its antimicrobial activity over usual aquaculture pathogens remains practically unexplored. In the present work, we have studied the effect of adding the terminal tag polyhistidine (6xHis) and the 2A peptide from the foot and mouth disease virus (FMDV-2A) to the N- or the C- terminus of caerin on its activity against a collection of bacterial and viral pathogens common to aquaculture species. In addition, the antimicrobial activity of a peptide composed of two tandem consecutive caerin sequences has been investigated. We have concluded that the addition of terminal tags to caerin causes a drastic reduction of its antiviral activity against the studied viruses and that the effect of the added tags on the bactericidal activity of the peptide depends of the target bacterial species. However, caerin with a 6xHis extension at its N terminus maintains or increases its antimicrobial activity against most of the fish pathogenic bacteria tested. According to our data, there is only a moderate agreement between the theoretical predictions obtained from in silico models and the experimental observations. This information is essential for the rational design of strategies to aim the recombinant production of caerin and other antimicrobial peptides, which can be used as anti-infective agents in aquaculture.

Targeted delivery of a short antimicrobial peptide against CD44-overexpressing tumor cells using hyaluronic acid-coated chitosan nanoparticles: An in vitro study

Studies have shown that cationic antimicrobial peptides can be effective against tumor cells. Accordingly, in the current study, we evaluated the antitumor activity of a short cationic antimicrobial peptide (CM11) against CD44 overexpressing tumor cell lines, A549, SH-SY5Y, and PANC-1, by developing a targeted delivery system based on hyaluronic acid–coated chitosan nanoparticles (HA-CS NPs) in vitro. For preparation of delivery system, CM11-loaded chitosan nanoparticles were fabricated by the ionic gelation method, and in order to bond the carboxylic group of hyaluronic acid to amine groups of chitosan, EDC as the coupling agent was used. Following the optimization, the particle size and zeta potential of HA-CS NPs were measured which were 190 nm and + 28.9 mv with reasonable PDI of 0.280. In addition, encapsulation efficiency of peptide in nanoparticles was estimated approximately 60%. The in vitro release rate of the peptide from nanoparticles was also pH-dependent, which was higher in acidic pH (5–6) compared with the physiological pH (7.4). Based on MTT assay, the cytotoxicity of CM11-loaded HA-CS NPs against tumor cell lines was significantly higher compared with the CM11-loaded CS NPs and free peptide, while the cytotoxic effect of the free peptide on the normal cells was more than the nanoparticles containing the peptide. Flow cytometry–based apoptosis/necrosis assay also exhibited CM11-NP–induced apoptotic cell death. Our findings confirm the significant potential of CM11 peptide against tumor cells and its delivery system in order to target these cells.

The effect of lipidation and glycosylation on short cationic antimicrobial peptides

The global health threat surrounding bacterial resistance has resulted in antibiotic researchers shifting their focus away from ‘traditional’ antibiotics and concentrating on other antimicrobial agents, including antimicrobial peptides. These low molecular weight "mini-proteins" exhibit broad-spectrum activity against bacteria, including multi-drug resistant strains, viruses, fungi and protozoa and constitute a major element of the innate-immune system of many multicellular organisms. Some naturally occurring antimicrobial peptides are lipidated and/or glycosylated and almost all antimicrobial peptides in clinical use are either lipopeptides (Daptomycin and Polymyxin E and B) or glycopeptides (Vancomycin). Lipidation, glycosylation and PEGylation are an option for improving stability and activity in serum and for reducing the rapid clearing via the kidneys and liver. Two broad-spectrum antimicrobial peptides NH2-RIRIRWIIR-CONH2 (A1) and NH2-KRRVRWIIW-CONH2 (B1) were conjugated via a linker, producing A2 and B2, to individual fatty acids of C8, C10, C12 and C14 and in addition, A2 was conjugated to either glucose, N-acetyl glucosamine, galactose, mannose, lactose or polyethylene glycol (PEG). Antimicrobial activity against two Gram-positive strains (methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE)) and three Gram-negative strains (Salmonella typhimurium, E. coli and Pseudomonas aeruginosa) were determined. Activity patterns for the lipidated versions are very complex, dependent on sequence, bacteria and fatty acid. Two reciprocal effects were measured; compared to the parental peptides, some combinations led to a 16-fold improvement whereas other combinations let to a 32-fold reduction in antimicrobial activity. Glycosylation decreased antimicrobial activity by 2 to 16-fold in comparison to A1, respectively on the sugar-peptide combination. PEGylation rendered the peptide inactive. Antimicrobial activity in the presence of 25% human serum of A1 and B1 was reduced 32-fold and 8-fold, respectively. The longer chain fatty acids almost completely restored this activity; however, these fatty acids increased hemolytic activity. B1 modified with C8 increased the therapeutic index by 2-fold for four bacterial strains. Our results suggest that finding the right lipid-peptide combination can lead to improved activity in the presence of serum and potentially more effective drug candidates for animal studies. Glycosylation with the optimal sugar and numbers of sugars at the right peptide position could be an alternative route or could be used in addition to lipidation to counteract solubility and toxicity issues.

In vitro toxicity evaluation of short cationic antimicrobial peptide (CM11) on Blastocystis sp

Blastocystis infection accounts for one of the causes of gastrointestinal problems with the prevalence rate of 3–100% worldwide. There is a wide range of drugs examined for the treatment of infected patients, among them metronidazole (MTZ) has been introduced as one of the efficient drugs. Besides to the suitable clinical effects, the administration of MTZ has some reported side-effects which emphasize on the identification of putative alternates. To this end, we aimed to evaluate the cytotoxicity effect of a newly-introduced synthetic antimicrobial peptide (AMP) named CM11 on in vitro cultured Blastocystis. Our results exhibited that CM11 treatment affected the viability of parasites in two cultural conditions including culturing alone and in co-culture with the Caco-2 cell line. The time- and dose-dependent effect of CM11 was consistent with the effect of MTZ which was used as control positive. The highest toxicity effect of CM11 was observed at the concentration of 24 μg/ml, leading to 28.7% and 25% viable parasites after 24 h and 48 h incubation times, respectively. Interestingly, the disruption of the Blastocystis cell membrane could be observed in the treated parasites. Therefore, CM11 can be suggested as a potential treatment for Blastocystis-infected patients after further in vitro and in vivo assessments.

Short Cationic Peptide Derived from Archaea with Dual Antibacterial Properties and Anti-Infective Potential.

Bacterial biofilms and associated infections represent one of the biggest challenges in the clinic, and as an alternative to counter bacterial infections, antimicrobial peptides have attracted great attention in the past decade. Here, ten short cationic antimicrobial peptides were generated through a sliding-window strategy on the basis of the 19-amino acid residue peptide, derived from a Pyrobaculum aerophilum ribosomal protein. PaDBS1R6F10 exhibited anti-infective potential as it decreased the bacterial burden in murine Pseudomonas aeruginosa cutaneous infections by more than 1000-fold. Adverse cytotoxic and hemolytic effects were not detected against mammalian cells. The peptide demonstrated structural plasticity in terms of its secondary structure in the different environments tested. PaDBS1R6F10 represents a promising antimicrobial agent against bacteria infections, without harming human cells.

In Silico Structural Evaluation of Short Cationic Antimicrobial Peptides.

Cationic peptides with antimicrobial properties are ubiquitous in nature and have been studied for many years in an attempt to design novel antibiotics. However, very few molecules are used in the clinic so far, sometimes due to their complexity but, mostly, as a consequence of the unfavorable pharmacokinetic profile associated with peptides. The aim of this work is to investigate cationic peptides in order to identify common structural features which could be useful for the design of small peptides or peptido-mimetics with improved drug-like properties and activity against Gram negative bacteria. Two sets of cationic peptides (AMPs) with known antimicrobial activity have been investigated. The first reference set comprised molecules with experimentally-known conformations available in the protein databank (PDB), and the second one was composed of short peptides active against Gram negative bacteria but with no significant structural information available. The predicted structures of the peptides from the first set were in excellent agreement with those experimentally-observed, which allowed analysis of the structural features of the second group using computationally-derived conformations. The peptide conformations, either experimentally available or predicted, were clustered in an “all vs. all” fashion and the most populated clusters were then analyzed. It was confirmed that these peptides tend to assume an amphipathic conformation regardless of the environment. It was also observed that positively-charged amino acid residues can often be found next to aromatic residues. Finally, a protocol was evaluated for the investigation of the behavior of short cationic peptides in the presence of a membrane-like environment such as dodecylphosphocholine (DPC) micelles. The results presented herein introduce a promising approach to inform the design of novel short peptides with a potential antimicrobial activity.

Control of green and blue mold and sour rot in citrus fruits by the cationic antimicrobial peptide PAF56

The ability of the short cationic antimicrobial peptide PAF56 (amino acid sequence GHRKKWFW) to control postharvest infectious diseases of citrus fruits was evaluated. PAF56 inhibited the growth of Penicillium digitatum, Penicillium italicum and Geotrichum candidum. The minimum inhibitory concentrations (MICs) of PAF56 for the control of P. digitatum, P. italicum and G. candidum were 8, 16, and 8μM, respectively. The conidia of P. digitatum, P. italicum and G. candidum were also highly sensitive to PAF56. The peptide PAF56 could effectively control green mold, blue mold and sour rot on inoculated citrus fruits. Fluorescence microscopy with the fluorescent stains SYTOX Green (SG) and Calcofluor White (CFW) was used to characterize the modes of action and interaction of PAF56 with the mycelia of P. digitatum, P. italicum and G. candidum. SG and CFW signals indicated that PAF56 could inhibit the formation of spores and change the cell membrane permeability and cell wall structure. A range of concentrations of PAF56 (from 16 to 128μM) showed no hemolysis or toxicity to human red blood cells (erythrocytes).

Dose-Response Effects of the CM11 as a Short Cationic Antimicrobial Peptide on Histopathological and Biochemical Changes in Mice

Dose-Response Effects of the CM11 as a Short Cationic Antimicrobial Peptide on Histopathological and Biochemical Changes in Mice

Cathelicidins Inhibit Escherichia coli-Induced TLR2 and TLR4 Activation in a Viability-Dependent Manner.

Activation of the immune system needs to be tightly regulated to provide protection against infections and, at the same time, to prevent excessive inflammation to limit collateral damage to the host. This tight regulation includes regulating the activation of TLRs, which are key players in the recognition of invading microbes. A group of short cationic antimicrobial peptides, called cathelicidins, have previously been shown to modulate TLR activation by synthetic or purified TLR ligands and may play an important role in the regulation of inflammation during infections. However, little is known about how these cathelicidins affect TLR activation in the context of complete and viable bacteria. In this article, we show that chicken cathelicidin-2 kills Escherichia coli in an immunogenically silent fashion. Our results show that chicken cathelicidin-2 kills E. coli by permeabilizing the bacterial inner membrane and subsequently binds the outer membrane-derived lipoproteins and LPS to inhibit TLR2 and TLR4 activation, respectively. In addition, other cathelicidins, including human, mouse, pig, and dog cathelicidins, which lack antimicrobial activity under cell culture conditions, only inhibit macrophage activation by nonviable E. coli In total, this study shows that cathelicidins do not affect immune activation by viable bacteria and only inhibit inflammation when bacterial viability is lost. Therefore, cathelicidins provide a novel mechanism by which the immune system can discriminate between viable and nonviable Gram-negative bacteria to tune the immune response, thereby limiting collateral damage to the host and the risk for sepsis.

Mussel-Inspired Multifunctional Hydrogel Coating for Prevention of Infections and Enhanced Osteogenesis.

Prevention of postsurgery infection and promotion of biointegration are the key factors to achieve long-term success in orthopedic implants. Localized delivery of antibiotics and bioactive molecules by the implant surface serves as a promising approach toward these goals. However, previously reported methods for surface functionalization of the titanium alloy implants to load bioactive ingredients suffer from time-consuming complex processes and lack of long-term stability. Here, we present the design and characterization of an adhesive, osteoconductive, and antimicrobial hydrogel coating for Ti implants. To form this multifunctional hydrogel, a photo-cross-linkable gelatin-based hydrogel was modified with catechol motifs to enhance adhesion to Ti surfaces and thus promote coating stability. To induce antimicrobial and osteoconductive properties, a short cationic antimicrobial peptide (AMP) and synthetic silicate nanoparticles (SNs) were introduced into the hydrogel formulation. The controlled release of AMP loaded in the hydrogel demonstrated excellent antimicrobial activity to prevent biofilm formation. Moreover, the addition of SNs to the hydrogel formulation enhanced osteogenesis when cultured with human mesenchymal stem cells, suggesting the potential to promote new bone formation in the surrounding tissues. Considering the unique features of our implant hydrogel coating, including high adhesion, antimicrobial capability, and the ability to induce osteogenesis, it is believed that our design provides a useful alternative method for bone implant surface modification and functionalization.

Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin.

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G1 LLKR5 IKT8 LL-NH2 , it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly1 , Arg5 , and Thr8 and lipophilic groups with different lengths in the N-terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF5 IKK8 LL-NH2 exhibited high activity against Gram-negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N-terminus increased the antimicrobial activity against Gram-positive and Gram-negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α-helical conformation inducing better antimicrobial effects. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Insights into Mechanistic and Synergistic Aspects of Novel Synthetic Short Cationic Antibacterial Peptides

AbstractTwo series of short cationic antimicrobial peptides (CAMPs) in which modification of the imidazole ring of histidine by bulkier alkyl substituents was done using regiospecific and radical‐mediated alkylation. The synthesized CAMPs were evaluated for their antibacterial efficacy using different bacterial strains. In particular, CAMP 11 i was found to be most active against L. monocytogenes and other CAMPs such as 10 e and 11 g exhibited moderate activity against S. aureus.The most active compounds were tested against Hek‐293 and HeLa cells with active CAMPs 11 i and 11 g with calculation of selectivity index against the L. monocytogenesand S. aureus, respectively as compared to mammalian cells. The SEM studies were also performed which confirmed the disruption of cell wall of treated bacteria using CAMP 11 i at its MIC concentration. The selectivity of active peptides towards bacterial cells in comparison to mammalian cells was checked using tryptophan quenching studies on small unilamellar vesicles. The results were found to be perfectly in corroboration with the differences between bacterial and mammalian cell membrane composition, thereby, indicating that these peptides kill the bacterial cells via conjugation with the cell membrane. Synergy studies of CAMPs in combination with known clinical antibacterial drugs against L. monocytogenes further displayed enhanced antibacterial efficacy.

Short Cationic Antimicrobial Peptides Display Superior Antifungal Activities toward Candidiasis and Onychomycosis in Comparison with Terbinafine and Amorolfine.

Novel antifungals are in high demand due to the challenges associated with resistant, persistent, and systemic fungal infections. Synthetic mimics of antimicrobial peptides are emerging as a promising class of compounds for antifungal treatment. In the current study, five synthetic cationic antimicrobial tripeptides were evaluated as antifungal therapeutics against 24 pathogenic strains of fungi. Three of the peptides displayed strong general antifungal properties at low micromolar inhibitory concentrations. The most promising peptide, compound 5, was selected and evaluated as an antifungal remedy for Candida albicans candidiasis in a human skin model and for the treatment of Trichophyton rubrum induced onychomycosis in an infected human nail model. Compound 5 was shown to display antifungal properties and a rapid mode of action superior to those of both the commercial comparators Loceryl and Lamisil. Compound 5 was also active against a clinical isolate of Candida albicans with acquired fluconazole resistance.

Short native α-helical cationic antimicrobial peptides: promising alternative antibiotics

Short native α-helical cationic antimicrobial peptides: promising alternative antibiotics

Synthetic cationic antimicrobial peptides bind with their hydrophobic parts to drug site II of human serum albumin

BackgroundMany biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics.ResultsThe binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the μM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs.ConclusionsThe biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful consideration in antimicrobial peptide studies, as the systemic distribution can be significantly affected by HSA interactions.