A common landscape for membrane‐active peptides

Three families of membrane‐active peptides are commonly found in nature and are classified according to their initial apparent activity. Antimicrobial peptides are ancient components of the innate immune system and typically act by disruption of microbial membranes leading to cell death. Amyloid peptides contribute to the pathology of diverse diseases from Alzheimer's to type II diabetes. Preamyloid states of these peptides can act as toxins by binding to and permeabilizing cellular membranes. Cell‐penetrating peptides are natural or engineered short sequences that can spontaneously translocate across a membrane. Despite these differences in classification, many similarities in sequence, structure, and activity suggest that peptides from all three classes act through a small, common set of physical principles. Namely, these peptides alter the Brownian properties of phospholipid bilayers, enhancing the sampling of intrinsic fluctuations that include membrane defects. A complete energy landscape for such systems can be described by the innate membrane properties, differential partition, and the associated kinetics of peptides dividing between surface and defect regions of the bilayer. The goal of this review is to argue that the activities of these membrane‐active families of peptides simply represent different facets of what is a shared energy landscape.     

Bioactive conformations of two seminal delta opioid receptor penta‐peptides inferred from free‐energy profiles

Delta‐opioid (DOP) receptors are members of the G protein‐coupled receptor (GPCR) sub‐family of opioid receptors, and are evolutionarily related, with homology exceeding 70%, to cognate mu‐opioid (MOP), kappa‐opioid (KOP), and nociceptin opioid (NOP) receptors. DOP receptors are considered attractive drug targets for pain management because agonists at these receptors are reported to exhibit strong antinociceptive activity with relatively few side effects. Among the most potent analgesics targeting the DOP receptor are the linear and cyclic enkephalin analogs known as DADLE (Tyr‐D ‐Ala‐Gly‐Phe‐D ‐Leu) and DPDPE (Tyr‐D ‐Pen‐Gly‐Phe‐D ‐Pen), respectively. Several computational and experimental studies have been carried out over the years to characterize the conformational profile of these penta‐peptides with the ultimate goal of designing potent peptidomimetic agonists for the DOP receptor. The computational studies published to date, however, have investigated only a limited range of timescales and used over‐simplified representations of the solvent environment. We provide here a thorough exploration of the conformational space of DADLE and DPDPE in an explicit solvent, using microsecond‐scale molecular dynamics and bias‐exchange metadynamics simulations. Free‐energy profiles derived from these simulations point to a small number of DADLE and DPDPE conformational minima in solution, which are separated by relatively small energy barriers. Candidate bioactive forms of these peptides are selected from identified common spatial arrangements of key pharmacophoric points within all sampled conformations. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 21–27, 2014.     

Peptide array‐based characterization and design of ZnO‐high affinity peptides

Peptides with both an affinity for ZnO and the ability to generate ZnO nanoparticles have attracted attention for the self‐assembly and templating of nanoscale building blocks under ambient conditions with compositional uniformity. In this study, we have analyzed the specific binding sites of the ZnO‐binding peptide, EAHVMHKVAPRP, which was identified using a phage display peptide library. The peptide binding assay against ZnO nanoparticles was performed using peptides synthesized on a cellulose membrane using the spot method. Using randomized rotation of amino acids in the ZnO‐binding peptide, 125 spot‐synthesized peptides were assayed. The peptide binding activity against ZnO nanoparticles varied greatly. This indicates that ZnO binding does not depend on total hydrophobicity or other physical parameters of these peptides, but rather that ZnO recognizes the specific amino acid alignment of these peptides. In addition, several peptides were found to show higher binding ability compared with that of the original peptides. Identification of important binding sites in the EAHVMHKVAPRP peptide was investigated by shortened, stepwise sequence from both termini. Interestingly, two ZnO‐binding sites were found as 6‐mer peptides: HVMHKV and HKVAPR. The peptides identified by amino acid substitution of HKVAPR were found to show high affinity and specificity for ZnO nanoparticles. Biotechnol. Bioeng. 2010;106: 845–851. © 2010 Wiley Periodicals, Inc.     

Structural requirements for cellular uptake of α‐helical amphipathic peptides

The structure of the cell‐permeable α‐helical amphipathic model peptide FLUOS‐KLALKLALKALKAALKLA‐NH₂ ( I ) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 μm . These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non‐amphipathic 18‐mer peptides with different primary structure, net charge and helix parameters from I . The amphipathic counterparts were internalized into the cells to a comparable extent as I , whereas no cellular uptake could be detected for the non‐amphipathic analogues. The mode of uptake remains unclear and involves both temperature‐sensitive and ‐insensitive processes, indicating non‐endocytic contributions. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Peptide internalization enabled by folding: triple helical cell‐penetrating peptides

Cell‐penetrating peptides (CPPs) are known as efficient transporters of molecular cargo across cellular membranes. Their properties make them ideal candidates for in vivo applications. However, challenges in the development of effective CPPs still exist: CPPs are often fast degraded by proteases and large concentration of CPPs required for cargo transporting can cause cytotoxicity. It was previously shown that restricting peptide flexibility can improve peptide stability against enzymatic degradation and limiting length of CPP peptide can lower cytotoxic effects. Here, we present peptides (30‐mers) that efficiently penetrate cellular membranes by combining very short CPP sequences and collagen‐like folding domains. The CPP domains are hexa‐arginine (R₆) or arginine/glycine (RRGRRG). Folding is achieved through multiple proline–hydroxyproline–glycine (POG [proline‐hydroxyproline‐glycine])_n repeats that form a collagen‐like triple helical conformation. The folded peptides with CPP domains are efficiently internalized, show stability against enzymatic degradation in human serum and have minimal toxicity. Peptides lacking correct folding (random coil) or CPP domains are unable to cross cellular membranes. These features make triple helical cell‐penetrating peptides promising candidates for efficient transporters of molecular cargo across cellular membranes. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Translocation of cell‐penetrating peptides into Candida fungal pathogens

Cell‐penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF)₃K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration‐dependent manner, and an additional peptide (TP‐10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF)₃K, and TP‐10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens.     

Cystine‐knot peptides targeting cancer‐relevant human cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4)

Cystine‐knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine‐knot peptide MCoTI‐II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine‐knot trypsin inhibitor into a CTLA‐4 binder by screening a library of variants using yeast surface display. A set of cystine‐knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400‐fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis,characterization, and evaluation of novel cell‐penetrating peptides based on TD‐34

In this study, six N‐1, N‐2, or N‐11 derivatives of TD‐34 (a cationic cyclic cell‐penetrating peptide [CPP], ACSSKKSKHCG) were designed and synthesized including both linear peptides and cyclic peptides, such as DL‐1 (KWSSKKSKHCG), DLCC‐1 (cyclopeptide, KWSSKKSKHCG), DL‐2 (KWSSKKSKHCG‐NH₂), DLCC‐2 (cyclopeptide, KWSSKKSKHCG‐NH₂), DL‐3 (RWSSKKSKHCG), and DLCC‐3 (cyclopeptide, RWSSKKSKHCG). The cyclic peptides were synthesized by disulfide bound linkages formed by N‐2 and N‐10 cysteine. In vitro penetration experiment was conducted to investigate the transdermal enhancement ability of these derivatives, using triptolide (TP) as model drug. The results display that at the presence of DLCC‐2, the accumulative penetration amount of TP increased 1.71‐fold (P < .05) within 12 hours, displaying better transdermal enhancing ability than TD‐34. Meanwhile, DL‐3 and DLCC‐3 slightly decreased the transdermal delivery of TP, and the presence of DL‐1 and DLCC‐1 shows no obvious effect. In order to clarify the factors on the transdermal ability of peptides, the solubility of TP in phosphate buffer saline (PBS) at the presence of different peptides and the mechanism of transdermal delivery of CPPs was investigated. The result shows that most of these peptides have no significant effect on the solubility of TP except DLCC‐3 (the solubility of TP slightly increased). And in order to investigate transdermal absorption route of DLCC‐2, polyarginine linked to rhodamine b (Rh b) derivative is used. The result proved that the transdermal route of polyarginine is via hair follicle, which may change the transdermal route of its cargo molecule (TP). Our group previously proved that polyarginine and TD‐34 have similar transdermal enhancing mechanism (changing the transdermal route of their cargo molecule); it is reasonably speculated that the transdermal route of DLCC‐2 is the same as polyarginine and then changes the transdermal absorption route of TP. Furthermore, such results have laid a solid foundation for further investigation of CPPs and paved a way for both designing and synthesizing of new drug delivery system for therapy molecules.     

Anti‐biofilm and sporicidal activity of peptides based on wheat puroindoline and barley hordoindoline proteins

The broad‐spectrum activity of antimicrobial peptides (AMPs) and low probability of development of host resistance make them excellent candidates as novel bio‐control agents. A number of AMPs are found to be cationic, and a small proportion of these are tryptophan‐rich. The puroindolines (PIN) are small, basic proteins found in wheat grains with proposed roles in biotic defence of seeds and seedlings. Synthetic peptides based on their unique tryptophan‐rich domain (TRD) display antimicrobial properties. Bacterial endospores and biofilms are highly resistant cells, with significant implications in both medical and food industries. In this study, the cationic PIN TRD‐based peptides PuroA (FPVTWRWWKWWKG‐NH₂) and Pina‐M (FSVTWRWWKWWKG‐NH₂) and the related barley hordoindoline (HIN) based Hina (FPVTWRWWTWWKG‐NH₂) were tested for effects on planktonic cells and biofilms of the common human pathogens including Pseudomonas aeruginosa, Listeria monocytogenes and the non‐pathogenic Listeria innocua. All peptides showed significant bactericidal activity. Further, PuroA and Pina‐M at 2 × MIC prevented initial biomass attachment by 85–90% and inhibited >90% of 6‐h preformed biofilms of all three organisms. However Hina, with a substitution of Lys‐9 with uncharged Thr, particularly inhibited Listeria biofilms. The PIN based peptides were also tested against vegetative cells and endospores of Bacillus subtilis. The results provided evidence that these tryptophan‐rich peptides could kill B. subtilis even in sporulated state, reducing the number of viable spores by 4 log units. The treated spores appeared withered under scanning electron microscopy. The results establish the potential of these tryptophan‐rich peptides in controlling persistent pathogens of relevance to food industries and human health. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Self‐assembling amyloid‐like peptides as exogenous second harmonic probes for bioimaging applications

Amyloid‐like peptides are an ideal model for the mechanistic study of amyloidosis, which may lead to many human diseases, such as Alzheimer disease. This study reports a strong second harmonic generation (SHG) effect of amyloid‐like peptides, having a signal equivalent to or even higher than those of endogenous collagen fibers. Several amyloid‐like peptides (both synthetic and natural) were examined under SHG microscopy and shown they are SHG‐active. These peptides can also be observed inside cells (in vitro). This interesting property can make these amyloid‐like peptides second harmonic probes for bioimaging applications. Furthermore, SHG microscopy can provide a simple and label‐free approach to detect amyloidosis. Lattice corneal dystrophy was chosen as a model disease of amyloidosis. Morphological difference between normal and diseased human corneal biopsy samples can be easily recognized, proving that SHG can be a useful tool for disease diagnosis.     

Studies of insect peptides alloferon,Any‐GS and their analogues. Synthesis and antiherpes activity

The subject of these studies was synthesis and determination of biological properties of a series of insect peptides, such as alloferon, Any‐GS and their analogues. The synthesis of 14 peptides was performed by the solid‐phase method. Biological effect of these peptides was evaluated by the antiviral test against Human Herpes Virus type 1 (HHV‐1) in vitro using a Vero cell line. It was found that the investigated peptides inhibit the replication of HHV‐1 in Vero cells. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Cecropin D‐like antibacterial peptides from the sphingid moth,Agrius convolvuli

Two major antibacterial peptides were isolated and purified from immunized larval hemolymph of Agrius convolvuli. Acid extraction, gel filtration, ultrafiltration, and reversed‐phase FPLC were used for purification of peptides. These peptides had similar molecular mass and amino acid composition. Moreover, 21 of the first 23 N terminal residues were identical. The peptides were highly homologous with cecropin D in size and primary sequence, and named Agrius cecropin D1 and D2. The molecular masses of Agrius cecropin D1 and D2 were 3,879.39 and 3,839.27, respectively. In antibacterial and hemolytic assays, Agrius cecropin D showed potent antibacterial activities against a panel of Gram positive and negative bacteria without hemolytic activity against human red blood cells. Notably, our antibacterial assay revealed Agrius cecropin D possessed stronger or at least equivalent activities against B. megaterium than cecropin A. It suggests that Agrius cecropin D, which has an alternative structure from cecropin D, could be the model for the development of peptide antibiotics. Arch. Insect Biochem. Physiol. 41:178–185, 1999. © 1999 Wiley‐Liss, Inc.     

Short PlGF‐derived peptides bind VEGFR‐1 and VEGFR‐2 in vitro and on the surface of endothelial cells

The placental growth factor (PlGF), a member of VEGF family, plays a crucial role in pathological angiogenesis, especially ischemia, inflammation, and cancer. This activity is mediated by its selective binding to VEGF receptor 1 (VEGFR‐1), which occurs predominantly through receptor domains 2 and 3. The PlGF β‐hairpin region spanning residues Q87 to V100 is one of the key binding elements on the protein side. We have undertaken a study on the design, preparation, and functional characterization of the peptide reproducing this region and of a set of analogues where glycine 94, occurring at the corner of the hairpin in the native protein, is replaced by charged as well as hydrophobic residues. Also, some peptides with arginine 96 replaced by other residues have been studied. We find that the parent peptide weakly binds VEGFR‐1, but replacement of G94 with residues bearing H‐bond donating residues significantly improves the affinity. Replacement of R96 instead blocks the interaction between the peptide and the domain. The strongest affinity is observed with the G94H (peptide PlGF‐2) and G94W (peptide PlGF‐10) mutants, while the peptide PlGF‐8, bearing the R96G mutation, is totally inactive. The PlGF‐1 and PlGF‐2 peptides also bind the VEGFR‐2 receptors, though with a reduced affinity, and are able to interfere with the VEGF‐induced receptor signaling on endothelial cells. The peptides also bind VEGFR‐2 on the surface of cells, while PlGF‐8 is inactive. Data suggest that these peptides have potential applications as PlGF/VEGF mimic in various experimental settings.     

Improved anticancer potency by head‐to‐tail cyclization of short cationic anticancer peptides containing a lipophilic β2,2‐amino acid

We have recently reported a series of synthetic anticancer heptapeptides (H‐KKWβ^2,2WKK‐NH₂) containing a central achiral and lipophilic β^2,2‐amino acid that display low toxicity against non‐malignant cells and high proteolytic stability. In the present study, we have further investigated the effects of increasing the rigidity and amphipathicity of two of our lead heptapeptides by preparing a series of seven to five residue cyclic peptides containing the two most promising β^2,2‐amino acid derivatives as part of the central lipophilic core. The peptides were tested for anticancer activity against human Burkitt's lymphoma (Ramos cells), haemolytic activity against human red blood cells (RBC) and cytotoxicity against healthy human lung fibroblast cells (MRC‐5). The results demonstrated a considerable increase in anticancer potency following head‐to‐tail peptide cyclization, especially for the shortest derivatives lacking a tryptophan residue. High‐resolution NMR studies and molecular dynamics simulations together with an annexin‐V‐FITC and propidium iodide fluorescent assay showed that the peptides had a membrane disruptive mode of action and that the more potent peptides penetrated deeper into the lipid bilayer. The need for new anticancer drugs with novel modes of action is demanding, and development of short cyclic anticancer peptides with an overall rigidified and amphipathic structure is a promising approach to new anticancer agents. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Antifungal peptides: a potential new class of antifungals for treating vulvovaginal candidiasis caused by fluconazole‐resistant Candida albicans

Vulvovaginal candidiasis/candidosis is a common fungal infection afflicting approximately 75% of women globally caused primarily by the yeast Candida albicans. Fluconazole is widely regarded as the antifungal drug of choice since its introduction in 1990 due to its high oral bioavailability, convenient dosing regimen and favourable safety profile. However, its widespread use has led to the emergence of fluconazole‐resistant C. albicans, posing a universal clinical concern. Coupled to the dearth of new antifungal drugs entering the market, it is imperative to introduce new drug classes to counter this threat. Antimicrobial peptides (AMPs) are potential candidates due to their membrane‐disrupting mechanism of action. By specifically targeting fungal membranes and being rapidly fungicidal, they can reduce the chances of resistance development and treatment duration. Towards this goal, we conducted a head‐to‐head comparison of 61 short linear AMPs from the literature to identify the peptide with the most potent activity against fluconazole‐resistant C. albicans. The 11‐residue peptide, P11‐6, was identified and assayed against a panel of clinical C. albicans isolates followed by fungicidal/static determination and a time‐kill assay to gauge its potential for further drug development. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.