Effect of hydrophobic modifications in antimicrobial peptides

With increasing resistance development against conventional antibiotics, there is an urgent need to identify novel approaches for infection treatment. Antimicrobial peptides may offer opportunities in this context, hence there has been considerable interest in identification and optimization of such peptides during the last decade in particular, with the long-term aim of developing these to potent and safe therapeutics. In the present overview, focus is placed on hydrophobic modifications of antimicrobial peptides, and how these may provide opportunities to combat also more demanding pathogens, including multi-resistant strains, yet not provoking unacceptable toxic responses. In doing so, physicochemical factors affecting peptide interactions with bacterial and eukaryotic cell membranes are discussed. Throughout, an attempt is made to illustrate how physicochemical studies on model lipid membranes can be correlated to result from bacterial and cell assays, and knowledge from this translated into therapeutic considerations.     

Skin and wound delivery systems for antimicrobial peptides

Non-healing wounds cause hundreds of thousands of deaths every year, and result in large costs for society. A key reason for this is the prevalence of challenging bacterial infections, which may dramatically hinder wound healing. With resistance development among bacteria against antibiotics, this situation has deteriorated during the last couple of decades, pointing to an urgent need for new wound treatments. In particular, this applies to wound dressings able to combat bacterial infection locally in wounds and impaired skin, including those formed by bacteria resistant to conventional antibiotics. Within this context, antimicrobial peptides (AMPs) are currently receiving intense interest. AMPs are amphiphilic peptides, frequently net positively charged, and with a sizable fraction of hydrophobic amino acids. Through destabilization of bacterial membranes, neutralization of inflammatory lipopolysaccharides, and other mechanisms, AMPs can be designed for potent antimicrobial effects, also against antibiotics-resistant strains, and to provide immunomodulatory effects while simultaneously displaying low toxicity. While considerable attention has been placed on AMP optimization and clarification of their mode(s)-of-action, much less attention has been paid on efficient AMP delivery. Considering that AMPs are large molecules, net positively charged, amphiphilic, and susceptible to infection-mediated proteolytic degradation, efficient in vivo delivery of such peptides is, however, challenging and delivery systems needed for the realization of AMP-based therapeutics. In the present work, recent developments regarding AMP delivery systems for treatment of wounds and skin infections are discussed, with the aim to link results from physicochemical studies on, e.g., peptide loading/release, membrane interactions, and self-assembly, with those on the biological functional performance of AMP delivery systems in terms of antimicrobial effects, cell toxicity, inflammation, and wound healing.     

Interplay between amphiphilic peptides and nanoparticles for selective membrane destabilization and antimicrobial effects

As a result of an increasing number of bacteria developing resistance against antibiotics, antimicrobial peptides (AMPs) are attracting significant interest, particularly in relation to identification of peptides displaying potent but selective effects. Much less focus has been placed on delivery systems for AMPs, despite AMPs suffering from delivery challenges related to their size, cationicity, and amphiphilicity. Inorganic nanoparticles may provide opportunities for controlling peptide release, reducing infection-related AMP degradation, or increasing bioavailability. Numerous such nanomaterials display potent and triggerable antimicrobial effects on their own. When combined with AMPs, combinatorial and synergistic effects in relation to the behavior of such mixed systems as antimicrobials have been observed. The mechanistic origin of these effects are poorly understood that at present, however, precluding rational design of mixed nanoparticle antimicrobials/AMPs and nanoparticulate delivery systems for AMPs. Here, the area of membrane interactions and antimicrobial effects of inorganic nanomaterials are briefly outlined, in combination with AMPs.     

Recent advances in antimicrobial surfaces via tunable molecular interactions: Nanoarchitectonics and bioengineering applications

Infections can lead to severe health issues, even death. Surfaces, such as those of biomedical devices, implants, textiles, tables and doorknobs, play a crucial role as carriers for pathogens to migrate, attach and proliferate. Implementing surfaces with antimicrobial properties offers a reliable and long-lasting approach to combat surface transmission of germs, minimize microbial colonization, and reduce infections. In this review, we present recent advancements in antimicrobial surfaces, categorized into four groups based on their action mechanisms: antifouling, bactericidal, antifouling and bactericidal, and dynamic or stimuli-responsive surfaces. The work highlights the fabrication processes and properties of each category, along with discussing their structure-performance relationships. Special attention is given to various anchoring strategies involving tunable molecular interactions. The review also introduces relevant biomedical applications.     

新型抗菌肽的设计、活性研究及与磷脂相互作用的计算模拟

设计合成了多个具有2个活性序列的线性和环状多肽及具有单个活性序列的短链多肽, 研究了它们的杀菌活性, 发现其杀菌活性顺序为长链肽>环状肽>短链肽, 特别是线性的Linear-KT和Linear-KS对多种革兰氏阴性菌和阳性菌均具有较高的杀菌活性. 采用MTT法考察了Linear-KT和Linear-KS对正常细胞的毒性, 其中Linear-KS表现出较低的细胞毒性, 优于阳性对照多粘菌素B. 利用计算模拟的方法计算了多肽与细菌细胞膜中磷脂酰甘油(DMPG)的相互作用. 结果表明, 多肽和DMPG的结合能也表现出长链肽>环状肽>短链肽的规律, 特别是Linear-KT和Linear-KS具有较高的结合能. 长链肽含有2个活性序列, 可提供多个荷正电的氨基酸与荷负电的磷脂结合, 结合能较大, 杀菌活性较强. 同时, 柔性的结构及Linear-KT和Linear-KS中丝氨酸和苏氨酸的β碳上的羟基可与磷脂上的羰基形成多个氢键, 进一步增大了结合能. 计算模拟的方法为抗菌肽的杀菌活性从理论上提供了一定的依据.     

促红细胞生成素(EPO)受体(EBP)激活剂的理论突变设计

采用分子动力学模拟的方法, 构建了人促红细胞生成素模拟肽与其受体胞外结合片段的相互作用的分子动力学模型. 通过对该模型的结构研究和理论分析, 对模拟肽与受体结合机制提出了新的理论解释. 根据EBP活性口袋的静电势分布, 对二聚体小肽激活剂的每条链上的部分氨基酸进行了突变, 分别用电性更强的氨基酸来代替部分疏水氨基酸, 计算结果显示, 突变后的二聚体小肽激活剂对EBP的“亲和力”明显增强.     

Electrochemistry of peptides

This opinion is focused on the electrochemistry of peptides utilised in biological and technological processes. The redox behaviour of peptides important in biomedicine, neuropeptides and antimicrobial peptides is highlighted. In addition to peptides composed of essential amino acids, peptide-mimetic molecules such as peptide nucleic acid and artificial peptidic wires transferring protons and electrons are reviewed. The application of electrochemistry for the research of peptide–membrane and peptide–surface interactions is also discussed.     

The effects of solution structure on the surface conformation and orientation of a cysteine-terminated antimicrobial peptide cecropin P1

The surface structure of an antimicrobial peptide, cecropin P1, immobilized to a gold surface via a terminal cysteine residue was investigated. Using reflection-absorption infrared spectroscopy, surface plasmon resonance, and X-ray photoelectron spectroscopy, the effects of pH, solution conformation, and concentration on the immobilized peptide conformation, average orientation, and surface density were determined. Under all conditions investigated, the immobilized peptides were α-helical in a predominately flat, random orientation. The addition of the reducing agent Tris(2-carboxyethyl) phosphine hydrochloride to the buffer resulted in a twofold increase in immobilized peptide surface density.     

Assessment of conformation and energetics of the N-terminal part of elafin via computer simulations

Elafin, a specific inhibitor of elastase, is thought to play a regulatory role in inflammation. An NMR-derived solution structure of recombinant elafin has been reported [Francart et al. (1997) J Mol Biol 268:666 ], although the conformation of its flexible N-terminal part is not established. There is experimental evidence that the N terminus (residues 1–15) of elafin interacts with the cell membrane. To explore the conformational preferences of residues in this region, we have performed Monte Carlo simulations of the peptide in water, in cyclohexane, and in a model membrane. Additionally, 3.7-ns molecular dynamics with explicit water was carried out. The main results were that the hydrophobic environment stabilizes an α helix in the region 6–11, the peptide is unordered in water, and it is attached to the membrane via the amphiphilic α-helix 6–11, which inserts with its N terminus forming an angle of about 60° to the membrane plane. We therefore assume that in nonpolar media the N-terminal part of elafin forms a short α helix which might act as a membrane anchor. Received: 5 July 2000 / Accepted: 4 October 2000 / Published online: 28 February 2001     

Rational design of hirulog-type inhibitors of thrombin

Summary The two crystal structures of thrombin complexed with its most potent natural inhibitor hirudin and with the active-site inhibitor d-Phe-Pro-Arg-CH₂Cl [Rydel, T.J. et al., J. Mol. Biol., 221 (1991) 583; Bode, W. et al., EMBO J., 8 (1989) 3467] were used as a basis to design a new inhibitor, combining the high specificity of the polypeptide hirudin with the simpler chemistry of an organic compound. In the new inhibitor, the C-terminal amino acid residues 53–65 of hirudin are linked by a spacer peptide of four glycines to the active-site inhibitor NAPAP (N-(2-naphthyl-sulfonyl-glycyl)-dl-p-amidinophenylalanyl-piperidine). Energy minimization techniques served as a tool to determine the preferred configuration at the amidinophenylalanine and the modified piperidine moiety of the inhibitor. The predictions are supported by the interaction energies determined for d- and l-NAPAP in complex with thrombin, which are in good agreement with experimentally determined dissociation constants. The conformational flexibility of the linker peptide in the new inhibitors was investigated with molecular dynamics techniques. A correlation between the P1 position and the interactions of the linker peptide with the protein is suggested. Modifications of the linker peptide are proposed based on the distribution of its main-chain torsion angles in order to enhance its binding to thrombin.     

A biofilm resistance surface yielded by grafting of antimicrobial peptides on stainless steel surface

Anti‐biofilm formation on the surface is a severe issue in medical implants, hull surface, and food industry. Antimicrobial peptide, magainin II, was covalently bound to stainless steel surfaces through multi‐step modification. The untreated and modified samples were analyzed by SEM‐EDS, XPS, and contact angle, respectively, which indicated the peptide was immobilized on the surfaces. The antimicrobial tests of modified samples were conducted using Staphylococcus aureus and Escherichia coli, and the results revealed that peptide modified surface decreased the biofilm and bacteria quantity of stainless steel surface.     

A chromatography-focused bioprocess that eliminates soluble aggregation for bioactive production of a new antimicrobial peptide candidate

Human beta defensins (hBDs) are an important class of antimicrobial peptides (AMPs), which provide the host with innate protection from bacteria, fungi and viruses. Human β-defensin-25 (hBD25) is a new hBD variant which has been recently discovered in the male genital tract. Since its discovery, hBD25 was hypothesized to play a key role in protection against genital tract infection, which has significantly increased mortality rates in the last decade. However, further studies to confirm the role of hBD25 are hindered by the lack of sufficient amounts of pure hBD25 for clinical studies. This study reports the first successful development of an efficient and low cost chromatography-oriented bioprocess for production of hBD25. hBD25 was expressed predominantly as soluble aggregates although the peptide was co-expressed with a Maltose Binding Protein (MBP) fusion tag in E. coli. The soluble aggregates were disrupted by denaturation-reduction of the hBD25, followed by an in vitro size exclusion chromatography refolding step which readily yielded bioactive and purified hBD25 peptides at 90% purity. The refolded hBD25 showed antimicrobial activity against E. coli K12 at a minimal inhibitory concentration of 60 μg/mL. With an overall hBD25 bioprocess yield of 48% obtained, this bioprocess will open the way for detailed clinical studies of hBD25, and serve as a generic platform for efficient recovery of other 'fusion protein'-derived peptides that inevitably exist as soluble aggregates.     

Molecular docking and dynamic simulation of approved drugs targeting against spike protein (6VXX) of 2019-nCoV (novel coronavirus)

The 2019-nCoV has triggered a global public health emergency due to its rapid spread, resulting in a pandemic situation. Because of its ability to bind with the host cell receptor ACE-2, the spike protein of the 2019-nCoV is a critical factor in viral infection. The current study aims to investigate the molecular-docking of the spike protein (6VXX) using PyRx for FDA-approved drugs available for the treatment of SARS-1 and MERS, with the hypothesis that these drugs could be suggested for the treatment of 2019-nCoV or not. A phylogenetic analysis of 2019-nCoV in relation to SARS-1 and MERS confirmed the validation. The positive result urged the Multiple Sequence Alignment analysis of the top five affected countries, with China serving as a control, using WHO available reference data to determine the rate of mutant variation. The docking results revealed that the top ten drugs with the highest binding affinity rate are also used for Hepatitis-C virus treatment, and the Molecular Dynamic Simulation was carried out for the drug Paritaprevir, which had the highest binding affinity rate, using Gromacs. The results indicated that the drug Paritaprevir could be used as a potential target against the 2019-nCoV Spike protein.     

Antioxidant and antimicrobial study of Schefflera vinosa leaves crude extracts against rice pathogens

Plant extracts are one of the best possible sources of bioactive molecules, and are being used globally as an antioxidants and natural antimicrobial compounds. In current study, Schefflera vinosa leaves extract was prepared through Soxhlet extraction procedure using methanol and chloroform as solvents. The extract was investigated for total antioxidant, phenolic and flavonoid contents, free radical scavenging and antimicrobial activities. The free radical scavenging activities were evaluated through 2,2- diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-3-ethylbenzotiazolin-6-sulfonic acid (ABTS) and Ferric-reducing/ antioxidant power (FRAP) assay. The antimicrobial activity of extract was determined through poisoned food method. The methanolic extract has exhibited high antioxidant, phenolic, and flavonoid activities compared to chloroform extract. Similarly, free radical scavenging activities (ABTS, DPPH and FRAP) were higher in methanolic extract. Further, Fourier-Transform Infrared Spectroscopy (FTIR) used to determine the functional group and Gas chromatography-mass spectrometry (GC–MS) to elucidate volatile composition of the crude extract. Different functional group like N-H, O-H, C-O, C-N, C-H, C=O, C≡C and C-O-H presence indicate the existence of many metabolites in the extracts. GC–MS study identified 61 compounds and subsequently, these molecules were screened virtually using DockThor. Furthermore, antimicrobial study was confirmed against rice pathogens like Magnaporthe oryzae (M. oryzae) and Xanthomonas oryzae pv. oryzae (Xoo). Molecular docking study further suggested that phytomolecules (3-Isopropoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tris (trimethylsiloxy) tetrasiloxane, and 2-Methoxy-5-methylthiophene) targets Histone Deacetylase (HDAC) of M. oryzae and Peptide Deformylase (PDF) of Xoo, which could inhibit their growth. Hence, this study indicated that Schefflera vinosa extracts could be an important ingredient as an antioxidant as well as antimicrobial agent against rice pathogens.     

High-level expression of acidic partner-mediated antimicrobial peptide from tandem genes inEscherichia coli

A novel strategy for constructing multiple joined genes of acidic partner-mediated antimicrobial peptide is described. This strategy allows the expression of antimicrobial peptide byEscherichia coli in a stable form and with high yield. Cecropin A (1–8)-melittin (1–10) (CAME) hybrid peptide was selected as a model of antimicrobial peptide. An acidic fragment from magainin intervening sequence was fused to the antimicrobial peptide as a partner to neutralize the lethal effects on the host cells. Multiple copies of the fusion peptide gene were tandemly linked and cloned into the expression vector pET21a. Multimers were expressed at high levels, reaching up to 36% of total cell proteins, and expression levels were proportional to the degree of multimerization. The fusion proteins were mainly expressed as inclusion bodies, probably owing to cysteine residues in the multimers. The target CAME peptide was obtained by cleaving the multimers with cyanogen bromide and purified by cation-exchange chromatography. Recombinant CAME peptide showed strong antimicrobial activities against both Gram-negative and -positive bacteria. These results might provide an efficient solution for high-level expression of various kinds of antimicrobial peptides that are toxic to the host.     

Estimation of acidity constants,ionic mobilities and charges of antimicrobial peptides by capillary electrophoresis

Capillary electrophoresis (CE) was employed for the determination of thermodynamic acidity constants (pK_a) and actual ionic mobilities of polycationic antimicrobial peptides (AMPs). The effective electrophoretic mobilities of AMPs were measured by CE in a series of the background electrolytes within a wide pH range (2.00–12.25), at constant ionic strength (25 mM) and ambient temperature, using polybrene coated fused silica capillaries to suppress sorption of cationic AMPs to the capillary wall. Eventually, Haarhoff–Van der Linde peak fitting function was used for the determination of correct migration times of some AMPs peaks that were distorted by electromigration dispersion. The measured effective mobilities were corrected to 25°C. Mixed acidity constants, , and actual ionic mobilities, m_i, of AMPs were determined by the nonlinear regression analysis of pH dependence of their effective mobilities. The values were recalculated to thermodynamic pK_as using the Debye–Hückel theory. Thermodynamic pK_a of imidazolium group of histidine residues was found to be in the range 3.72–4.98, pK_a of α‐NH₃⁺ group was in the range 6.14–6.93, and pK_a of ε‐NH₃⁺ group of lysine spanned the interval 7.26–9.84, depending on the particular amino acid sequence of the AMPs. Actual ionic mobilities of AMPs with positive charges from one to six elementary units achieved values (9.8 – 36.5) × 10^?9 m²V^?1s^?1.     

Rational in silico design of aptamers for organophosphates based on the example of paraoxon

Poisoning by organophosphates (OPs) takes one of the leading places in the total number of exotoxicoses. Detoxication of OPs at the first stage of the poison entering the body could be achieved with the help of DNA- or RNA-aptamers, which are able to bind poisons in the bloodstream. The aim of the research was to develop an approach to rational in silico design of aptamers for OPs based on the example of paraoxon. From the published sequence of an aptamer binding organophosphorus pesticides, its three-dimensional model has been constructed. The most probable binding site for paraoxon was determined by molecular docking and molecular dynamics (MD) methods. Then the nucleotides of the binding site were mutated consequently and the values of free binding energy have been calculated using MD trajectories and MM-PBSA approach. On the basis of the energy values, two sequences that bind paraoxon most efficiently have been selected. The value of free binding energy of paraoxon with peripheral anionic site of acetylcholinesterase (AChE) has been calculated as well. It has been revealed that the aptamers found bind paraoxon more effectively than AChE. The peculiarities of paraoxon interaction with the aptamers nucleotides have been analyzed. The possibility of improving in silico approach for aptamer selection is discussed.     

Rational Design of a DNA-Scaffolded High-Affinity Binder for Langerin

Binders of langerin could target vaccines to Langerhans cells for improved therapeutic effect. Since langerin has low affinity for monovalent glycan ligands, highly multivalent presentation has previously been key for targeting. Aiming to reduce the amount of ligand required, we rationally designed molecularly defined high-affinity binders based on the precise display of glycomimetic ligands (Glc2NTs) on DNA-PNA scaffolds. Rather than mimicking langerin's homotrimeric structure with a C3-symmetric scaffold, we developed readily accessible, easy-to-design bivalent binders. The method considers the requirements for bridging sugar binding sites and statistical rebinding as a means to both strengthen the interactions at single binding sites and amplify the avidity enhancement provided by chelation. This gave a 1150-fold net improvement over the affinity of the free ligand and provided a nanomolar binder (IC₅₀=300 nM) for specific internalization by langerin-expressing cells.     

Assessment of antimicrobial and enzymes inhibition effects of Allium kastambulense with in silico studies: Analysis of its phenolic compounds and flavonoid contents

Type 2 diabetes and obesity-related metabolic diseases have been treated with traditional medicinal plants for centuries. In this study, the effects of Allium kastambulense plant extracts on different enzyme activities were investigated, and the results were added as graphics and tables after calculating. This study aimed to identify and quantify the phenolic composition of Allium kastambulense Bosse and determine the anti-lipase, anti-urease, anti-melanogenesis, antidiabetic, anti-alzheimer, and antibacterial properties. IC₅₀ results for all enzymes were obtained between 0.55 and 138 µg/mL, and this plant inhibited HMG_CoA R and tyrosinase enzymes more with IC₅₀ values of 0.55 and 59.17 µg/mL, respectively. The interactions of active compounds showing activity against different enzymes were examined with molecular docking studies. The most active compound 3, (rosmarinic acid) has ?10.90 kcal/mol binding energy value against HMG_CoA R, and also the potential structure compound 2, (+catechin), which has activity against α-amylase, α-glycosidase, and lipase enzymes, was –8.30, ?8.40 and ?9.70 kcal/mol, respectively. Finally, antimicrobial effects, total phenolic, and flavonoid content, determined with its higher total phenolic (22.63 mg GAE/g extract) and flavonoid (6.41 mgQE/g extract) contents and main chemical compounds of this plant were gentisic acid, (+) catechin, and rosmarinic acid, respectively.