Important structural features of antimicrobial peptides towards specific activity: Trends in the development of efficient therapeutics.

Proteins and peptides, as polypeptide chains, have usually got unique conformational structures for effective biological activity. Antimicrobial peptides (AMPs) are a group of bioactive peptides, which have been increasingly studied during recent years for their promising antibacterial, antifungal, antiviral and anti-inflammatory activity, as well as, other esteemed bioactivities. Numerous AMPs have been separated from a wide range of natural resources, or produced in vitro through chemical synthesis and recombinant protein expression. Natural AMPs have had limited clinical application due to several drawbacks, such as their short half-life due to protease degradation, lack of activity at physiological salt concentrations, toxicity to mammalian cells, and the absence of suitable methods of delivery for the AMPs that are targeted and sustained. The creation of synthetic analogs of AMPs would both avoid the drawbacks of the natural analogs and maintain or even increase the antimicrobial effectiveness. The structure-activity relationship of discovered AMPs or their derivatives facilitates the development of synthetic AMPs. This review discovered that the relationship between the activity of AMPs and their positive net charge, hydrophobicity, and amino acid sequence and the relationship between AMPs' function and other features like their topology, glycosylation, and halogenation.

Rational Design of Amphipathic Antimicrobial Peptides with Alternating L-/D-Amino Acids That Form Helical Structures.

Antimicrobial peptides (AMPs) are promising therapeutic agents against bacteria. We have previously reported an amphipathic AMP Stripe composed of cationic L-Lys and hydrophobic L-Leu/L-Ala residues, and Stripe exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria. Gramicidin A (GA), composed of repeating sequences of L- and D-amino acids, has a unique ß6.3-helix structure and exhibits broad antimicrobial activity. Inspired by the structural properties and antimicrobial activities of LD-alternating peptides such as GA, in this study, we designed Stripe derivatives with LD-alternating sequences. We found that simply alternating L- and D-amino acids in the Stripe sequence to give StripeLD caused a reduction in antimicrobial activity. In contrast, AltStripeLD, with cationic and hydrophobic amino acids rearranged to yield an amphipathic distribution when the peptide adopts a ß6.3-helix, displayed higher antimicrobial activity than AltStripe. These results suggest that alternating L-/D-cationic and L-/D-hydrophobic amino acids in accordance with the helical structure of an AMP may be a useful way to improve antimicrobial activity and develop new AMP drugs.

Designing the antimicrobial peptide with centrosymmetric and amphipathic characterizations for improving antimicrobial activity.

Antibiotic-resistant bacterial infections are becoming a serious health issue and will cause 10 million deaths per year by 2050. As a result, the development of new antimicrobial agents is urgently needed. Antimicrobial peptides (AMPs) are found in the innate immune systems of various organisms to effectively fend off invading pathogens. In this study, we designed a series of AMPs (THL-2-1 to THL-2-9) with centrosymmetric and amphipathic properties, through substituting different amino acids on the hydrophobic side and at the centrosymmetric position to improve their antimicrobial activity. The results showed that leucine as a residue on the hydrophobic side of the peptide could enhance its antimicrobial activity and that glutamic acid as a centrosymmetric residue could increase the salt resistance of the peptide. Thus, the THL-2-3 peptide (KRLLRELKRLL-NH2 ) showed the greatest antimicrobial activity (MIC90 of 16 µM) against Gram-negative bacteria and had the highest salt resistance and cell selectivity among all the designed peptides. In summary, the results of this study provide useful references for the design of AMPs to enhance antimicrobial activity.

Structure-activity relationship study of amphipathic antimicrobial peptides using helix-destabilizing sarcosine.

Antimicrobial peptides (AMPs) are potential therapeutic agents against bacteria. We recently showed that a rationally designed AMP, termed Stripe, with an amphipathic distribution of native cationic and hydrophobic amino acids on its helical structure exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria with negligible hemolytic activity and cytotoxicity. In this study, the structure-activity relationship of Stripe was elucidated by designing a series of antimicrobial peptides whereby amino acid residues of Stripe were exchanged with helix-destabilizing sarcosine residues. Stripe 1-5 peptides with hydrophobic amino acids substituted with sarcosine were predominantly unstructured and showed no antimicrobial activity, except against Escherichia coli (E. coli) (DH5α) cells. The activity against E. coli (DH5α) cells and the helicity of Stripe 1-5 peptides decreased concomitantly as the number of sarcosine residue substitutions increased. Stripe 1-5 peptides showed no hemolytic activity or cytotoxicity. The results indicate that sarcosine substitutions provide an approach to study the structure-activity relationship of helical AMPs, and the helicity of Stripe is an important feature defining its activity.

Development of Antimicrobial Stapled Peptides Based on Magainin 2 Sequence.

Magainin 2 (Mag2), which was isolated from the skin of the African clawed frog, is a representative antimicrobial peptide (AMP) that exerts antimicrobial activity via microbial membrane disruption. It has been reported that the helicity and amphipathicity of Mag2 play important roles in its antimicrobial activity. We investigated and recently reported that 17 amino acid residues of Mag2 are required for its antimicrobial activity, and accordingly developed antimicrobial foldamers containing α,α-disubstituted amino acid residues. In this study, we further designed and synthesized a set of Mag2 derivatives bearing the hydrocarbon stapling side chain for helix stabilization. The preferred secondary structures, antimicrobial activities, and cell-membrane disruption activities of the synthesized peptides were evaluated. Our analyses revealed that hydrocarbon stapling strongly stabilized the helical structure of the peptides and enhanced their antimicrobial activity. Moreover, peptide 2 stapling between the first and fifth position from the N-terminus showed higher antimicrobial activity than that of Mag2 against both gram-positive and gram-negative bacteria without exerting significant hemolytic activity. To investigate the modes of action of tested peptides 2 and 8 in antimicrobial and hemolytic activity, electrophysiological measurements were performed.

Rational design, conformational analysis and membrane-penetrating dynamics study of Bac2A-derived antimicrobial peptides against gram-positive clinical strains isolated from pyemia.

The Bac2A (RLARIVVIRVAR-NH2) is a linearized counterpart of cationic cyclic peptide Bactenecin-one of the smallest naturally occurring antimicrobial peptides (AMPs), which, however, generally exhibits a low or moderate antibacterial potency against gram-positive bacteria. Here, it is found that the Bac2A and its linear derivates cannot spontaneously fold into a well-defined helical conformation in solution, thus impairing the peptide amphipathicity and antibacterial activity. Hydrocarbon stapling is rationally designed to constrain the helical conformation of these linear peptides. Atomistic dynamics simulations reveal that the membrane-penetrating course of linear and stapled peptides include four distinct phases, during which the stapled peptides can maintain in an ordered helical conformation, while linear peptides are structured from intrinsic disorder in water solution to partially helical state in membrane interior, indicating that lipid environment can help the linear peptide refolding into amphipathic helix, although the refolding process would incur a large configurational entropy penalty. The antibacterial activities of the most potent stapled peptide are determined as MIC = 7.6 and 16 µg/ml against two gram-positive Staphylococcus aureus clinical strains isolated from pyemia. The activity values are improved by 7.1-fold and 5-fold as compared to that of native Bac2A peptide with MIC = 54 and 80 µg/ml, respectively.

Selectivity of Antimicrobial Peptides: A Complex Interplay of Multiple Equilibria.

Antimicrobial peptides (AMPs) attack bacterial membranes selectively, killing microbes at concentrations that cause no toxicity to the host cells. This selectivity is not due to interaction with specific receptors but is determined by the different lipid compositions of the membranes of the two cell types and by the peculiar physicochemical properties of AMPs, particularly their cationic and amphipathic character. However, the available data, including recent studies of peptide-cell association, indicate that this picture is excessively simplistic, because selectivity is modulated by a complex interplay of several interconnected phenomena. For instance, conformational transitions and self-assembly equilibria modulate the effective peptide hydrophobicity, the electrostatic and hydrophobic contributions to the membrane-binding driving force are nonadditive, and kinetic processes can play an important role in selective bacterial killing in the presence of host cells. All these phenomena and their bearing on the final activity and toxicity of AMPs must be considered in the definition of design principles to optimize peptide selectivity.

Structural insight into the mechanism of action of antimicrobial peptide BMAP-28(1-18) and its analogue mutBMAP18.

Structural characterization of BMAP-28(1-18), a potent bovine myeloid antimicrobial peptide can aid in understanding its mechanism of action at molecular level. We report NMR structure of the BMAP-28(1-18) and its mutated analogue mutBMAP18 in SDS micelles. Structural comparison of the peptides bound to SDS micelles and POPE-POPG vesicles using circular dichroism, suggest that structures in the two lipid preparations are similar. Antimicrobial assays show that even though both these peptides adopt helical conformation, BMAP-28(1-18) is more potent than mutBMAP18 in killing bacterial cells. Our EM images clearly indicate that the peptides target the bacterial cell membrane resulting in leakage of its contents. The structural basis for difference in activity between these peptides was investigated by molecular dynamics simulations. Inability of the mutBMAP18 to retain its helical structure in presence of POPE:POPG membrane as opposed to the BMAP-28(1-18) at identical peptide/lipid ratios could be responsible for its decreased activity. Residues Ser5, Arg8 and Arg12 of the BMAP-28(1-18) are crucial for its initial anchoring to the bilayer. We conclude that along with amphipathicity, a stable secondary structure that can promote/initiate membrane anchoring is key in determining membrane destabilization potential of these AMPs. Our findings are a step towards understanding the role of specific residues in antimicrobial activity of BMAP-28(1-18), which will facilitate design of smaller, cost-effective therapeutics and would also help prediction algorithms to expedite screening out variants of the parent peptide with greater accuracy.

Inhibition of polyglutamine aggregation by SIMILAR huntingtin N-terminal sequences: Prospective molecules for preclinical evaluation in Huntington's disease.

The mutant huntingtin protein (mHtt) fragments with expanded polyglutamine sequence forms microscopically visible aggregates in neurons, a hallmark of Huntington's disease (HD). The aggregation process and aggregates are possible targets of therapeutic intervention in HD. Owing to the lack of treatment and cure, the patients die within 15-20 years after the disease onset. Therefore, discovering therapeutic molecules that may either inhibit the aggregation mechanism or downregulate the toxic effects of mHtt are highly needed. This study demonstrates the design and use of peptide inhibitors based on the role played by the N-terminal seventeen amino acid sequence (NT17 ) of huntingtin fragment in its aggregation. Fug-NT17 (Fugu), Xen-NT17 (Xenopus), Dro-NT17 (Drosophila), Aib-NT17 , and Pro-NT17 sequences were tested for their ability to inhibit aggregation. Among them, the first three are the sequence variants of human NT17 from evolutionarily distant organisms and the latter two are the analogs of human NT17 -containing aminoisobutyric acid (Aib) and proline (Pro). Four out of five inhibited the aggregation of huntingtin fragment, NT17 Q35 P10 K2 polypeptide. Data indicate that the physicochemical properties of the inhibitors play a crucial role in exhibiting the inhibitory effect. These inhibitors can be tested in cell and animal models for the preclinical evaluation in the treating of HD.

Pro-apoptotic cationic host defense peptides rich in lysine or arginine to reverse drug resistance by disrupting tumor cell membrane.

Host defense peptides have been demonstrated to exhibit prominent advantages in cancer therapy with selective binding ability toward tumor cells via electrostatic attractions, which can overcome the limitations of traditional chemotherapy drugs, such as toxicity on non-malignant cells and the emergence of drug resistance. In this work, we redesigned and constructed a series of cationic peptides by inserting hydrophobic residues into hydrophilic surface or replacing lysine (K) with arginine (R), based on the experience from the preliminary work of host defense peptide B1. In-depth studies demonstrated that the engineered peptides exhibited more potent anti-cancer activity against various cancer cell lines and much lower toxicity to normal cells compared with B1. Further investigation revealed that compounds I-3 and I-7 could act on cancer cell membranes and subsequently alter the permeability, which facilitated obvious pro-apoptotic activity in paclitaxel-resistant cell line (MCF-7/Taxol). The result of mitochondrial membrane potential assay (ΔΨm) demonstrated that the peptides induced ΔΨm dissipation and mitochondrial depolarization. The caspase-3 cellular activity assay showed that the anti-cancer activity of peptides functioned via caspase-3-dependent apoptosis. The study yielded compound I-7 with superior properties for antineoplastic activity in comparison to B1, which makes it a promising potential candidate for cancer therapy.

Large-Scale Analysis of Antimicrobial Activities in Relation to Amphipathicity and Charge Reveals Novel Characterization of Antimicrobial Peptides.

It has been unclear to which antimicrobial activities (e.g., anti-gram-positive bacterial, anti-gram-negative bacterial, antifungal, antiparasitic, and antiviral activities) of antimicrobial peptides (AMPs) a given physiochemical property matters most. This is the first computational study using large-scale AMPs to examine the relationships between antimicrobial activities and two major physiochemical properties of AMPs-amphipathicity and net charge. The results showed that among all kinds of antimicrobial activities, amphipathicity and net charge best differentiated between AMPs with and without anti-gram-negative bacterial activities. In terms of amphipathicity and charge, all the AMPs whose activities were significantly associated with amphipathicity and net charge were alike except those with anti-gram-positive bacterial activities. Furthermore, the higher the amphipathic value, the greater the proportion of AMPs possessing both antibacterial and antifungal activities. This dose-response-like pattern suggests a possible causal relationship-dual antibacterial and antifungal activities of AMPs may be attributable to amphipathicity. These novel findings could be useful for identifying potent AMPs computationally.

Critical Evaluation and Compilation of Physicochemical Determinants and Membrane Interactions of MMGP1 Antifungal Peptide.

A growing issue of pathogen resistance to antibiotics has fostered the development of innovative approaches for novel drug development. Here, we report the physicochemical and biological properties of an antifungal peptide, MMGP1, based on computational analysis. Computation of physicochemical properties has revealed that the natural biological activities of MMGP1 are coordinated by its intrinsic properties such as net positive charge (+5.04), amphipathicity, high hydrophobicity, low hydrophobic moment, and higher isoelectric point (11.915). Prediction of aggregation hot spots in MMGP1 had revealed the presence of potentially aggregation-prone segments that can nucleate in vivo aggregation (on the membrane), whereas no aggregating regions were predicted for in vitro aggregation (in solutions) of MMGP1. This ability of MMGP1 to form oligomeric aggregates on membrane further substantiates its direct-cell penetrating potency. Monte Carlo simulation of the interactions of MMGP1 in the aqueous phase and different membrane environments revealed that increasing the proportion of acidic lipids on membrane had led to increase in the peptide helicity. Furthermore, the peptide adopts energetically favorable transmembrane configuration, by inserting peptide loop and helix termini into the membrane containing >60% of anionic lipids. The charged lipid-based insertion of MMGP1 into membrane might be responsible for the selectivity of peptide toward fungal cells. Additionally, MMGP1 possessed DNA-binding property. Computational docking has identified DNA-binding residues (TRP3, SER4, MET7, ARG8, PHE10, ALA11, GLY20, THR21, ARG22, MET23, TRP34, and LYS36) in MMGP1 crucial for its DNA-binding property. Furthermore, computational mutation analysis revealed that aromatic amino acids are crucial for in vivo aggregation, membrane insertion, and DNA-binding property of MMGP1. These data provide new insight into the molecular determinants of MMGP1 antifungal activity and also serves as the template for the design of novel peptide antibiotics.

Cyclic non-opioid dynorphin A analogues for the bradykinin receptors.

Nerve injury and inflammation cause up-regulation of an endogenous opioid ligand, dynorphin A (Dyn A), in the spinal cord resulting in hyperalgesia via the interaction with bradykinin receptors (BRs). This is a non-opioid neuroexcitatory effect that cannot be blocked by opioid antagonists. Our systematic structure-activity relationships study on Dyn A identified lead ligands 1 and 4, along with the key structural feature (i.e. amphipathicity) for the BRs. However, the ligands showed very low metabolic stability in plasma (t1/2 <1h) and therefore, in order to improve their metabolic stabilities with retained biological activities, various modifications were performed. Cyclization of ligand 4 afforded a cyclic Dyn A analogue 5 that retained the same range of binding affinity as the linear ligand with improved metabolic stability (t1/2 >5h) and therefore possesses the potential as a pharmacophoric scaffold to be utilized for drug development.

Modification of amphipathic non-opioid dynorphin A analogues for rat brain bradykinin receptors.

It has been shown that under chronic pain or nerve injury conditions, up-regulated dynorphin A (Dyn A) interacts with bradykinin receptors (BRs) to cause hyperalgesia in the spinal cord. Thus BRs antagonist can modulate hyperalgesia by blocking Dyn A's interaction with the BRs in the central nervous system. In our earlier structure-activity relationship (SAR) study, [des-Arg(7)]-Dyn A-(4-11) 13 was discovered as a minimum pharmacophore for rat brain BRs with its antagonist activity (anti-hyperalgesic effect) in in vivo tests using naïve or injured animals. We have pursued further modification on the [des-Arg(7)]-Dyn A analogues and identified a key insight into the pharmacophore of the rat brain BRs: amphipathicity.

Membrane Affinity of Platensimycin and Its Dialkylamine Analogs.

Membrane permeability is a desired property in drug design, but there have been difficulties in quantifying the direct drug partitioning into native membranes. Platensimycin (PL) is a new promising antibiotic whose biosynthetic production is costly. Six dialkylamine analogs of PL were synthesized with identical pharmacophores but different side chains; five of them were found inactive. To address the possibility that their activity is limited by the permeation step, we calculated polarity, measured surface activity and the ability to insert into the phospholipid monolayers. The partitioning of PL and the analogs into the cytoplasmic membrane of E. coli was assessed by activation curve shifts of a re-engineered mechanosensitive channel, MscS, in patch-clamp experiments. Despite predicted differences in polarity, the affinities to lipid monolayers and native membranes were comparable for most of the analogs. For PL and the di-myrtenyl analog QD-11, both carrying bulky sidechains, the affinity for the native membrane was lower than for monolayers (half-membranes), signifying that intercalation must overcome the lateral pressure of the bilayer. We conclude that the biological activity among the studied PL analogs is unlikely to be limited by their membrane permeability. We also discuss the capacity of endogenous tension-activated channels to detect asymmetric partitioning of exogenous substances into the native bacterial membrane and the different contributions to the thermodynamic force which drives permeation.

The amphipathic design in helical antimicrobial peptides.

Amphipathicity is a critical characteristic of helical antimicrobial peptides (AMPs). The hydrophilic region, primarily composed of cationic residues, plays a pivotal role in the initial binding to negatively charged components on bacterial membranes through electrostatic interactions. Subsequently, the hydrophobic region interacts with hydrophobic components, inducing membrane perturbation, ultimately leading to cell death, or inhibiting intracellular function. Due to the extensive diversity of natural and synthetic AMPs with regard to the design of amphipathicity, it is complicated to study the structure-activity relationships. Therefore, this work aims to categorize the common amphipathic design and investigate their impact on the biological properties of AMPs. Besides, the connection between current structural modification approaches and amphipathic styles was also discussed.

Phytic acid and graphene oxide functionalized sponge with special-wettability and electronegativity for oil-in-water emulsion separation in single-step.

Developing an emulsion separation material with one-step in-situ purifying capability and improved security in applications, especially for subsequent scale-up, is valuable but remains a challenge. Herein, the amphiphilic sponge (PA@RGO@MS) was prepared via impregnation and in-situ growth of the negatively charged hydrophilic phytic acid (PA) and the hydrophobic reduced graphene oxide (RGO) on the surface of the melamine sponge (MS) and applied in emulsion purification. The mechanics, wettability, absorption performance of the PA@RGO@MS were analyzed to identify its potential for stable demulsification. Results show that the PA@RGO@MS could purify emulsions (turbidity removal rate = 99.7%; TOC removal rate = 94.14%) in-situ in one step by simple shock absorption, profited from the hydrophilic and demulsification capability of PA, oil absorption of RGO, and wide reaction and storage space of MS. Targeting the emulsion with distinct properties (density, viscosity, and concentration) of the oil phase, the PA@RGO@MS could efficiently enable the purification. Meanwhile, the powerful flame-retardant granted from PA ensures the safe shipment and storage of sponges. The favorable cyclability (turbidity removal rate > 98.5% and TOC removal rate > 89.5% after 10 cycles) and diversified operating modes enhance the practical value of the PA@RGO@MS.

Coarse-Grain Simulations of Membrane-Adsorbed Helical Peptides.

The amphipathic α-helix is a common motif for peptide adsorption to membranes. Many physiologically relevant events involving membrane-adsorbed peptides occur over time and size scales readily accessible to coarse-grain molecular dynamics simulations. This methodological suitability, however, comes with a number of pitfalls. Here, I exemplify a multi-step adsorption equilibration procedure on the antimicrobial peptide Magainin 2. It involves careful control of peptide freedom to promote optimal membrane adsorption before other interactions are allowed. This shortens preparation times prior to production simulations while avoiding divergence into unrealistic or artifactual configurations.

Conventional and unconventional antimicrobials from fish, marine invertebrates and micro-algae.

All eukaryotic organisms, single-celled or multi-cellular, produce a diverse array of natural anti-infective agents that, in addition to conventional antimicrobial peptides, also include proteins and other molecules often not regarded as part of the innate defences. Examples range from histones, fatty acids, and other structural components of cells to pigments and regulatory proteins. These probably represent very ancient defence factors that have been re-used in new ways during evolution. This review discusses the nature, biological role in host protection and potential biotechnological uses of some of these compounds, focusing on those from fish, marine invertebrates and marine micro-algae.

Systematically Studying the Optimal Amino Acid Distribution Patterns of the Amphiphilic Structure by Using the Ultrashort Amphiphiles.

Amphipathicity has traditionally been considered to be essential for the de novo design or systematic optimization of antimicrobial peptides (AMPs). However, the current research methods to study the relationship between amphiphilicity and antimicrobial activity are inappropriate, because the key parameters (hydrophobicity, positive charge, etc.) and secondary structure of AMPs are changed. To systematically and accurately study the effects of amphiphilicity on antimicrobial properties of AMPs, we designed parallel series of AMPs with a different order of amino acids in a sequence composed only of Arg and either Trp (WR series) or Leu (LR series), under conditions in which other vital parameters were fixed. Furthermore, based on the WR and LR peptides that can form stable amphiphilic ß-sheet structures in the anionic membrane-mimetic environment, we found that high ß-sheet amphipathic was accompanied by strong antimicrobial activity. Of such peptides, W5 ([RW]4W) and L5 ([RL]4L) with a nicely amphipathic ß-sheet structure possessed the optimal therapeutic index. W5 and L5 also exhibited high stability in vitro and a potent membrane-disruptive mechanism. These results suggest that the alternate arrangement of hydrophobic and hydrophilic residues to form a stable amphipathic ß-sheet structure is an essential factor that significantly affects the antimicrobial properties.