Lipopeptides in microbial infection control: Scope and reality for industry

Lipopeptides are compounds that are formed by cyclic or short linear peptides linked with a lipid tail or other lipophilic molecules. Recently, several lipopeptides were characterized, showing surfactant, antimicrobial and cytotoxic activities. The properties of lipopeptides may lead to applications in diverse industrial fields including the pharmaceutical industry as conventional antibiotics; the cosmetic industry for dermatological product development due to surfactant and anti-wrinkle properties; in food production acting as emulsifiers in various foodstuffs; and also in the field of biotechnology as biosurfactants. Some lipopeptides have reached a commercial antibiotic status, such as daptomycin, caspofungin, micafungin, and anidulafungin. This will be the focus of this review. Moreover, the review presented here will focus on the biotechnological utilization of lipopeptides in different fields as well as the functional–structure relation, connecting recent aspects of synthesis and structure diversity.     

Characterization of a potent antimicrobial lipopeptide via coarse-grained molecular dynamics

The prevalence of antibiotic-resistant pathogens is a major medical concern, prompting increased interest in the development of novel antimicrobial compounds. One such set of naturally occurring compounds, known as antimicrobial peptides (AMPs), have broad-spectrum activity, but come with many limitations for clinical use. Recent work has resulted in a set of antimicrobial lipopeptides (AMLPs) with micromolar minimum inhibitory concentrations and excellent selectivity for bacterial membranes. To characterize a potent, synthetic lipopeptide, C16-KGGK, we used multi-microsecond coarse-grained simulations with the MARTINI forcefield, with a total simulation time of nearly 46μs. These simulations show rapid binding of C16-KGGK, which forms micelles in solution, to model bacterial lipid bilayers. Furthermore, upon binding to the surface of the bilayer, these lipopeptides alter the local lipid organization by recruiting negatively charged POPG lipids to the site of binding. It is likely that this drastic reorganization of the bilayer has major effects on bilayer dynamics and cellular processes that depend on specific bilayer compositions. By contrast, the simulations revealed no association between the lipopeptides and model mammalian bilayers. These simulations provide biophysical insights into lipopeptide selectivity and suggest a possible mechanism for antimicrobial action. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Connecting peptide physicochemical and antimicrobial properties by a rational prediction model

The increasing rate in antibiotic-resistant bacterial strains has become an imperative health issue. Thus, pharmaceutical industries have focussed their efforts to find new potent, non-toxic compounds to treat bacterial infections. Antimicrobial peptides (AMPs) are promising candidates in the fight against antibiotic-resistant pathogens due to their low toxicity, broad range of activity and unspecific mechanism of action. In this context, bioinformatics' strategies can inspire the design of new peptide leads with enhanced activity. Here, we describe an artificial neural network approach, based on the AMP's physicochemical characteristics, that is able not only to identify active peptides but also to assess its antimicrobial potency. The physicochemical properties considered are directly derived from the peptide sequence and comprise a complete set of parameters that accurately describe AMPs. Most interesting, the results obtained dovetail with a model for the AMP's mechanism of action that takes into account new concepts such as peptide aggregation. Moreover, this classification system displays high accuracy and is well correlated with the experimentally reported data. All together, these results suggest that the physicochemical properties of AMPs determine its action. In addition, we conclude that sequence derived parameters are enough to characterize antimicrobial peptides.     

Thermodynamics of Antimicrobial Lipopeptide Binding to Membranes: Origins of Affinity and Selectivity

The development of novel antibiotic drugs is one of the most pressing biomedical problems due to the increasing number of antibiotic-resistant pathogens. Antimicrobial peptides and lipopeptides are a promising category of candidates, but the molecular origins of their antimembrane activity is unclear. Here we explore a series of recently developed antimicrobial lipopeptides, using coarse-grained molecular-dynamics simulations and free energy methods to uncover the thermodynamics governing their binding to membranes. Specifically, we quantify C16-KGGK’s binding affinity to the two types of membrane by umbrella sampling. We also examined the origin of C16-KGGK’s selectivity for bacterial versus mammalian membranes by systematically varying the peptide sequence and salt concentration. Our data showed that the C16 hydrophobic tail is the main contributor to its affinity to lipid membrane, whereas the peptide portion is mainly responsible for its selectivity. Furthermore, the electrostatic interaction between the cationic peptide and anionic bacterial membrane plays a significant role in the selectivity.     

微生物脂肽的结构

根据结构特征,将脂肽分为环状脂肽和线形脂肽;按脂肪酸链和肽链的连接方式不同,进一步将环状脂肽分为3类。在此基础上,对脂肽的产生菌和脂肽结构进行了综述,并对脂肽的活性、作用机理及其应用进行了展望。     

Lipopeptide surfactants: Production,recovery and pore forming capacity

Lipopeptides are microbial surface active compounds produced by a wide variety of bacteria, fungi and yeast. They are characterized by highly structural diversity and have the ability to decrease the surface and interfacial tension at the surface and interface, respectively. Surfactin, iturin and fengycin of Bacillus subtilis are among the most studied lipopeptides. This review will present the main factors encountering lipopeptides production along with the techniques developed for their extraction and purification. Moreover, we will discuss their ability to form pores and destabilize biological membrane permitting their use as antimicrobial, hemolytic and antitumor agents. These open great potential applications in biomediacal, pharmaceutic and agriculture fields.     

Multiple Drug Targeting Potential of Novel Ligands Against Virulent Proteins of Candida albicans

Systemic candidiasis is one of the most common nosocomial systemic infections causing high mortality and morbidity. Although infections caused by other Candida species are increasing the majority of candidiasis is commonly caused by Candida albicans which accounts for 40–60% of the reported cases. Current antifungal treatment regime is feeble due to persistent multiple drug resistance. Moreover, antifungal agents are mostly synthetic drugs that cause toxic side effects on immune-compromised patients. So, not only finding novel drug candidates from natural sources is necessary but to identify such compounds that have multiple targeting potential is essential. Hence, in this study, we propose to screen and identify bioactive natural compounds based on flavonoids, terpenes, and alkaloids for their potential antifungal activity. This in silico study was carried out by targeting 16 major virulent protein targets of C. albicans with 91 ligands. We have reported few ligands having multi-targeting potential and further analyzed theirs in silico pharmacokinetic profile. In silico ADMET analysis and protein–protein interaction can not only help in refining better drug candidates but also shortlist ideal protein for drug targeting.

     

枯草芽孢杆菌JA脂肽类及挥发性物质抑菌效应的研究

枯草芽孢杆菌JA产生的脂肽类抗生素对植物病原真菌有广谱抗性。将发酵液经过酸沉淀、甲醇抽提以及反相高效液相色谱等步骤, 分离得到脂肽类抗生素的纯品。经IC50实验和抗菌谱测定, 考察了脂肽类抗生素对多种植物病原菌的作用, 确定了脂肽类抗生素的抗菌谱。深入研究表明, 枯草芽孢杆菌JA还产生未知成分的挥发性抑菌物质, 能够抑制灰霉病菌孢子的萌发和菌丝的生长。脂肽类抗生素和挥发性抑菌物质的协同作用, 有助于提高枯草芽孢杆菌的生物防治效果。     

枯草芽孢杆菌JA脂肽类及挥发性物质抑菌效应的研究

枯草芽孢杆菌JA产生的脂肽类抗生素对植物病原真菌有广谱抗性.将发酵液经过酸沉淀、甲醇抽提以及反相高效液相色谱等步骤,分离得到脂肽类抗生素的纯品.经IC50实验和抗菌谱测定,考察了脂肽类抗生素对多种植物病原菌的作用,确定了脂肽类抗生素的抗菌谱.深入研究表明,枯草芽孢杆菌JA还产生未知成分的挥发性抑菌物质,能够抑制灰霉病菌孢子的萌发和菌丝的生长.脂肽类抗生素和挥发性抑菌物质的协同作用,有助于提高枯草芽孢杆菌的生物防治效果.     

Polyvalency: a promising strategy for drug design

The simultaneous binding of multiple ligands on one entity to multiple receptors on another can result in an affinity that is significantly greater than that for the binding of a single ligand to a single receptor. This concept of "polyvalency" can be used to design molecules that are potent inhibitors of toxins and pathogens. We describe the design of potent polyvalent inhibitors that neutralize anthrax toxin in vivo as well as our attempts to elucidate the relationship between inhibitor structure and activity. We also highlight promising future avenues for research in polyvalent drug design.     

Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: Comparison with cationic antimicrobial peptides and lipopeptides

With the steady rise in the number of antibiotic-resistant Gram-positive pathogens, it has become increasingly important to find new antibacterial agents which are highly active and have novel and diversified mechanisms of action. Two classes will be discussed here: the cationic antimicrobial peptides, which are amphiphilic in nature, targeting membranes and increasing their permeability; and lipopeptides, which consist of linear or cyclic peptides with an N-terminus that is acylated with a fatty acid side chain. One member of the cyclic lipopeptide family, the anionic molecule daptomycin, has been extensively studied and is the major focus of this review. Models will be presented on its mode of action and comparisons will be made to the known modes of action of cationic antimicrobial peptides and other lipopeptides.     

Mode of action of the new antibiotic for Gram-positive pathogens daptomycin: comparison with cationic antimicrobial peptides and lipopeptides

With the steady rise in the number of antibiotic-resistant Gram-positive pathogens, it has become increasingly important to find new antibacterial agents which are highly active and have novel and diversified mechanisms of action. Two classes will be discussed here: the cationic antimicrobial peptides, which are amphiphilic in nature, targeting membranes and increasing their permeability; and lipopeptides, which consist of linear or cyclic peptides with an N-terminus that is acylated with a fatty acid side chain. One member of the cyclic lipopeptide family, the anionic molecule daptomycin, has been extensively studied and is the major focus of this review. Models will be presented on its mode of action and comparisons will be made to the known modes of action of cationic antimicrobial peptides and other lipopeptides.     

Thermodynamics of Antimicrobial Lipopeptide Binding to Membranes: Origins of Affinity and Selectivity

The development of novel antibiotic drugs is one of the most pressing biomedical problems due to the increasing number of antibiotic-resistant pathogens. Antimicrobial peptides and lipopeptides are a promising category of candidates, but the molecular origins of their antimembrane activity is unclear. Here we explore a series of recently developed antimicrobial lipopeptides, using coarse-grained molecular-dynamics simulations and free energy methods to uncover the thermodynamics governing their binding to membranes. Specifically, we quantify C16-KGGK’s binding affinity to the two types of membrane by umbrella sampling. We also examined the origin of C16-KGGK’s selectivity for bacterial versus mammalian membranes by systematically varying the peptide sequence and salt concentration. Our data showed that the C16 hydrophobic tail is the main contributor to its affinity to lipid membrane, whereas the peptide portion is mainly responsible for its selectivity. Furthermore, the electrostatic interaction between the cationic peptide and anionic bacterial membrane plays a significant role in the selectivity.     

In Silico Design of Dual-Binding Site Anti-Cholinesterase Phytochemical Heterodimers as Treatment Options for Alzheimer’s Disease

The number of patients with neurodegenerative diseases, particularly Alzheimer’s disease (AD), continues to grow yearly. Cholinesterase inhibitors (ChEIs) represent the first-line symptomatic drug treatment for mild-to-moderate AD; however, there is an unmet need to produce ChEIs with improved efficacy and reduced side effects. Herein, phytochemicals with reported anti-acetylcholinesterase (AChE) activity were ranked in silico for their anti-AChE potential. Ligands with a similar or higher binding affinity to AChE than galantamine were then selected for the design of novel dual-binding site heterodimeric drugs. In silico molecular docking of heterodimers with the target enzymes, AChE and butyrylcholinesterase (BuChE), were performed, and anti-cholinesterase binding affinities were compared with donepezil. Drug-likeliness properties and toxicity of the heterodimers were assessed using the SwissADME and ProTox-II webservers. Nine phytochemicals displayed similar or higher binding affinities to AChE than galantamine: sanguinarine > huperzine A > chelerythrine > yohimbine > berberine > berberastine > naringenin > akuammicine > carvone. Eleven heterodimeric ligands were designed with phytochemicals separated by four- or five-carbon alkyl-linkers. All heterodimers were theoretically potent AChE and BuChE dual-binding site inhibitors, with the highest affinity achieved with huperzine-4C-naringenin, which displayed 34% and 26% improved affinity to AChE and BuChE, respectively, then the potent ChEI drug, donepezil. Computational pharmacokinetic and pharmacodynamic screening suggested that phytochemical heterodimers would display useful gastrointestinal absorption and with relatively low predicted toxicity. Collectively, the present study suggests that phytochemicals could be garnered for the provision of novel ChEIs with enhanced drug efficacy and low toxicity.     

Identification and natural functions of cyclic lipopeptides from Bacillus amyloliquefaciens An6

Lipopeptides constitute a structurally diverse group of metabolites produced by various bacterial and fungal genera. In the past decades, research on lipopeptides has been fueled by their surfactant activities. However, natural functions of lipopeptides compounds have received considerably less attention. The aim of this study was to isolate and identify the lipopeptides from Bacillus amyloliquefaciens An6, and further evaluate their biological activities. An6 lipopeptides were detected by PCR using degenerated primers and MALDI‐TOF‐MS. An6 strain was found to produce surfactin, fengycin, and bacillomycin. Following their purification, the in vitro antioxidant activity of An6 lipopeptides was studied through different assays. The scavenging effect on 1,1‐diphenyl‐2‐picrylhydrazyl radicals at a dosage of 0.75 mg/mL was 81%. Its reducing power was concentration‐dependant and reached a maximum of 1.07 at 2.5 mg/mL. Moreover, they showed a strong inhibition of β‐carotene bleaching. An6 lipopeptides mixture was also found to display significant antimicrobial activity against several Gram‐positive, Gram‐negative bacteria, and fungal strains. An6 lipopeptides were insensitive to proteolytic enzymes, stable between pH 4.0 and 12.0, and resistant to high temperature. Our results provided enough evidence proving that An6 lipopeptides could be used as functional‐food components.     

Advances in macrocyclic peptide-based antibiotics

Macrocyclic peptide-based natural products have provided powerful new antibiotic drugs, drug candidates, and scaffolds for medicinal chemists as a source of inspiration to design novel antibiotics. While most of those natural products are active mainly against Gram-positive pathogens, novel macrocyclic peptide-based compounds have recently been described, which exhibit potent and specific activity against some of the most problematic Gram-negative ESKAPE pathogens. This mini-review gives an up-date on recent developments.     

Biological and structural effects of the conjugation of an antimicrobial decapeptide with saturated,unsaturated, methoxylated and branched fatty acids

The increasing bacterial resistance against conventional antibiotics has led to the search for new antimicrobial drugs with different modes of action. Cationic antimicrobial peptides (AMPs) and lipopeptides are promising candidates to treat infections because they act on bacterial membranes causing rapid destruction of sensitive bacteria. In this study, a decapeptide named A2 (IKQVKKLFKK) was conjugated at the N‐terminus with saturated, unsaturated, methoxylated and methyl ‐branched fatty acids of different chain lengths (C8 – C20), the antimicrobial and structural properties of the lipopeptides being then investigated. The attachment of the fatty acid chain significantly improved the antimicrobial activity of A2 against bacteria, and so, endowed it with moderated antifungal activity against yeast strains belonging to genus Candida. Lipopeptides containing hydrocarbon chain lengths between C8 and C14 were the best antibacterial compounds (MIC = 0.7 to 5.8 μM), while the most active compounds against yeast were A2 conjugated with methoxylated and enoic fatty acids (11.1 to 83.3 μM). The improvement in antimicrobial activity was mainly related to the amphipathic secondary structure adopted by A2 lipopeptides in the presence of vesicles that mimic bacterial membranes. Peptide conjugation with long hydrocarbon chains (C12 or more), regardless of their structure, significantly increased toxicity towards eukaryotic cells, resulting in a loss of selectivity. These findings suggest that A2‐derived lipopeptides are potential good candidates for the treatment of infectious diseases caused by bacteria and opportunistic pathogenic yeast belonging to genus Candida. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Preparation and in vitro evaluation of novel lipopeptide transfection agents for efficient gene delivery

Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safety, stability, and suitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles to overcome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA binding at neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies. The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptide complex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexes formed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particle sizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion of histidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary for high transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Luc, mediated by all the lipopeptides, was much higher than poly(L-lysine) (PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branched polyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidine residue produced spontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved by incorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purified and offer new insights into the mechanism of action of transfection complexes.     

3D-QSAR studies of 2,2-diphenylpropionates to aid discovery of novel potent muscarinic antagonists

Muscarinic acetylcholine receptors (mAChRs) consisting of five known subtypes, are widely distributed in both central and peripheral nervous systems for regulation of a variety of critical functions. The present theoretical study describes correlations between experimental and calculated molecular properties of 15 α-substituted 2,2-diphenylpropionate antimuscarinics using quantum chemical and pharmacophore generation methods to characterize the drug mAChR properties and design new therapeutics. The calculated stereoelectronic properties, such as total energies, bond distances, valence angles, torsion angles, HOMO–LUMO energies, reactivity indices, vibrational frequencies of ether and carbonyl moieties, and nitrogen atom proton affinity were found to be well correlated when compared with experimentally determined inhibition constants from the literature using three muscarinic receptor assays: [³H]NMS receptor binding, α-amylase release from rat pancreas, and guinea pig ileum contraction. In silico predicted toxicity on rat oral LD₅₀ values correlated well with the [³H]NMS binding in N4TG1 cells and α-amylase release assays, but not the ileum contraction assay. Next, to explore the functional requirements for potent activity of the compounds, we developed a preliminary 3D pharmacophore model using the in silico techniques. The resulting model contained a hydrogen bond acceptor site on the carbonyl oxygen atom and a ring aromatic feature on one of the two aromatic rings in these compounds. This model was used as a template to search an in-house database for novel analogs. We found compounds equal in inhibition potency to atropine and, importantly, six not reported before as antimuscarinics. These results demonstrate that this QSAR approach not only provides a basis for understanding the molecular mechanism of action but a pharmacophore to aid in the discovery and design of novel potent muscarinic antagonists.     

Lipopolysaccharide binding protein binds to triacylated and diacylated lipopeptides and mediates innate immune responses

LPS binding protein (LBP) is an acute-phase protein synthesized predominantly in the liver of the mammalian host. It was first described to bind LPS of Gram-negative bacteria and transfer it via a CD14-enhanced mechanism to a receptor complex including TLR-4 and MD-2, initiating a signal transduction cascade leading to the release of proinflammatory cytokines. In recent studies, we found that LBP also mediates cytokine induction caused by compounds derived from Gram-positive bacteria, including lipoteichoic acid and peptidoglycan fragments. Lipoproteins and lipopeptides have repeatedly been shown to act as potent cytokine inducers, interacting with TLR-2, in synergy with TLR-1 or -6. In this study, we show that these compounds also interact with LBP and CD14. We used triacylated lipopeptides, corresponding to lipoproteins of Borrelia burgdorferi, mycobacteria, and Escherichia coli, as well as diacylated lipopeptides, corresponding to, e.g., 2-kDa macrophage activating lipopeptide of Mycoplasma spp. Activation of Chinese hamster ovary cells transfected with TLR-2 by both lipopeptides was enhanced by cotransfection of CD14. Responsiveness of human mononuclear cells to these compounds was greatly enhanced in the presence of human LBP. Binding of lipopeptides to LBP as well as competitive inhibition of this interaction by LPS was demonstrated in a microplate assay. Furthermore, we were able to show that LBP transfers lipopeptides to CD14 on human monocytes using FACS analysis. These results support that LBP is a pattern recognition receptor transferring a variety of bacterial ligands including the two major types of lipopeptides to CD14 present in different receptor complexes.