Is the Membrane Lipid Matrix a Key Target for Action of Pharmacologically Active Plant Saponins?

This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and triterpenes were tested. A systematic analysis of the effects of these chemicals on the physicochemical properties of the lipid bilayers and on the formation and functionality of the reconstituted ion channels induced by antimicrobial agents was performed. It was found that digitonin, tribulosin, and dioscin substantially reduced the boundary potential of the phosphatidylcholine membranes. We concluded that saponins might affect the membrane boundary potential by restructuring the membrane hydration layer. Moreover, an increase in the conductance and lifetime of gramicidin A channels in the presence of tribulosin was due to an alteration in the membrane dipole potential. Differential scanning microcalorimetry data indicated the key role of the sapogenin core structure (steroid or triterpenic) in affecting lipid melting and disordering. We showed that an alteration in pore forming activity of syringomycin E by dioscin might be due to amendments in the lipid packing. We also found that the ability of saponins to disengage the fluorescent marker calcein from lipid vesicles might be also determined by their ability to induce a positive curvature stress.

Therapeutic design of peptide modulators of protein-protein interactions in membranes.

Membrane proteins play the central roles in a variety of cellular processes, ranging from nutrient uptake and signalling, to cell-cell communication. Their biological functions are directly related to how they fold and assemble; defects often lead to disease. Protein-protein interactions (PPIs) within the membrane are therefore of great interest as therapeutic targets. Here we review the progress in the application of membrane-insertable peptides for the disruption or stabilization of membrane-based PPIs. We describe the design and preparation of transmembrane peptide mimics; and of several categories of peptidomimetics used for study, including d-enantiomers, non-natural amino acids, peptoids, and ß-peptides. Further aspects of the review describe modifications to membrane-insertable peptides, including lipidation and cyclization via hydrocarbon stapling. These approaches provide a pathway toward the development of metabolically stable, non-toxic, and efficacious peptide modulators of membrane-based PPIs. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 µM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity.

Lipid rafts, KCa/ClCa/Ca2+ channel complexes and EGFR signaling: Novel targets to reduce tumor development by lipids?

Membrane lipid rafts are distinct plasma membrane nanodomains that are enriched with cholesterol, sphingolipids and gangliosides, with occasional presence of saturated fatty acids and phospholipids containing saturated acyl chains. It is well known that they organize receptors (such as Epithelial Growth Factor Receptor), ion channels and their downstream acting molecules to regulate intracellular signaling pathways. Among them are Ca2+ signaling pathways, which are modified in tumor cells and inhibited upon membrane raft disruption. In addition to protein components, lipids from rafts also contribute to the organization and function of Ca2+ signaling microdomains. This article aims to focus on the lipid raft KCa/ClCa/Ca2+ channel complexes that regulate Ca2+ and EGFR signaling in cancer cells, and discusses the potential modification of these complexes by lipids as a novel therapeutic approach in tumor development. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

Physical behavior of KR-12 peptide on solid surfaces and Langmuir-Blodgett lipid films: Complementary approaches to its antimicrobial mode against S. aureus.

Biophysical characterization of antimicrobial peptides helps to understand the mechanistic aspects of their action. The physical behavior of the KR-12 antimicrobial peptide (e.g. orientation and changes in secondary structure), was analyzed after interactions with a Staphylococcus aureus membrane model and solid surfaces. We performed antimicrobial tests using Gram-positive S. aureus (ATCC 25923) bacteria. Moreover, Langmuir-Blodgett experiments showed that the synthetic peptide can disturb the lipidic membrane at a concentration lower than the Minimum Inhibitory Concentration, thus confirming that KR-12/lipid interactions are involved. Partially- and fully-deactivated KR-12 hybrid samples were obtained by physisorption and covalent immobilization in chitosan/silica and glyoxal-rich solid supports. The correlation of Langmuir-Blodgett data with the α-helix formation, followed by FTIR-ATR in a frozen-like state, and the antimicrobial activity showed the importance of these interactions and conformation changes on the first step action mode of this peptide. This is the first time that material science (immobilization in solid surfaces assisted by FTIR-ATR analysis in frozen-like state) and physical (Langmuir-Blodgett/Schaefer) approaches are combined for exploring mechanistic aspects of the primary action mode of the KR-12 antimicrobial peptide against S. aureus.

Effects of titanium dioxide and zinc oxide nano-materials on lipid order in model membranes.

Engineered nano-materials (ENM) have been reported to affect lipid membrane permeability in cell models, but a mechanistic understanding of how these materials interact with biological membranes has not been described. To assess mechanisms of permeability, liposomes composed of DOPC, DOPS, or POPC, with or without cholesterol, were used as model membranes for measuring ENM-induced changes to lipid order to improve our understanding of ENM effects on membrane permeability. Liposomes were treated with either titanium dioxide (TiO2) or zinc oxide (ZnO) ENM, and changes to lipid order were measured by time-resolved fluorescence anisotropy of a lipophilic probe, Di-4-ANEPPDHQ. Both ENM increased lipid order in two lipid models differing in headgroup charge. TiO2 increased lipid order of POPC liposomes (neutral charge), while ZnO acted primarily on DOPS liposomes (negative charge). Addition of cholesterol to these models significantly increased lipid order while in some cases attenuated ENM-induced changes to lipid order. To assess the ability of ENM to induce membrane permeability, liposomes composed of the above lipids were assayed for membrane permeability by calcein leakage in response to ENM. Both ENM caused a dose-dependent increase in permeability in all liposome models tested, and the addition of cholesterol to the liposome models neither blocked nor reduced calcein leakage. Together, these experiments show that ENM increased permeability of small molecules (calcein) from model liposomes, and that the magnitude of the effect of ENM on lipid order depended on ENM surface charge, lipid head group charge and the presence of cholesterol in the membrane.

Potential Drug Targets in Mycobacterial Cell Wall: Non-Lipid Perspective.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB), still remains a deadly disease worldwide. With prolonged usage of anti-TB drugs, the current therapeutic regimes are becoming ineffective, particularly due to emergence of drug resistance in MTB. Under such compelling circumstances, it is pertinent to look for new drug targets. The cell wall envelope of MTB is composed of unique lipids that are frequently targeted for anti-TB therapy. This is evident from the fact that most of the commonly used front line drugs (Isoniazid and Ethambutol) act on lipid machinery of MTB. Thus, despite the fact that much of the attention is towards understanding the MTB lipid biology, in search for identification of new drug targets, our knowledge of bacterial cell wall non-lipid components remains rudimentary and underappreciated. Better understanding of such components of mycobacterial cell structure will help in the identification of new drug targets that can be utilized on the persistent mycobacterium. This review at a common platform summarizes some of the non-lipid cell wall components in MTB that have potential to be exploited as future drug targets.

Targeting the Achilles Heel of Mosquito-Borne Viruses for Antiviral Therapy.

Mosquito-borne viruses encompass a wide range of pathogens, such as dengue and Zika viruses, that often cocirculate geographically. These viruses affect hundreds of millions of people worldwide, yet no clinically approved therapy is currently available for treating these viral infections. Thus, innovative therapies, especially inhibitors with broad antiviral activities against all these viruses, are urgently needed. While traditional therapeutic strategies mainly focus on inhibiting viral replication in a "one lock, one key" manner (e.g., viral protease and polymerase inhibitors), inhibitors targeting virions have recently emerged as a promising approach to achieve broad antiviral activities. Within this approach, Lipid Envelope Antiviral Disruption (LEAD) molecules were shown to broadly inhibit mosquito-borne viruses and other lipid membrane-enveloped viruses. Several LEAD molecules have been demonstrated to act against viral membranes in vitro, some of which have even shown in vivo efficacy to treat mosquito-borne viral infections. This therapeutic potential is further enhanced by molecular engineering to improve the inhibitors' pharmacological properties, laying the foundation for the LEAD antiviral strategy to be explored for possible treatment of mosquito-borne viral infections.

Membrane bound COMT isoform is an interfacial enzyme: general mechanism and new drug design paradigm.

The enzyme catechol-O-methyltransferase (COMT) has water soluble (S-COMT) and membrane associated (MB-COMT), bitopic, isoforms. Of these MB-COMT is a drug target in relation to the treatment of Parkinson's disease. Using a combination of computational and experimental protocols, we have determined the substrate selection mechanism specific to MB-COMT. We show: (1) substrates with preferred affinity for MB-COMT over S-COMT orient in the membrane in a fashion conducive to catalysis from the membrane surface and (2) binding of COMT to its cofactor ADOMET induces conformational change that drives the catalytic surface of the protein to the membrane surface, where the substrates and Mg2+ ions, required for catalysis, are found. Bioinformatics analysis reveals evidence of this mechanism in other proteins, including several existing drug targets. The development of new COMT inhibitors with preferential affinity for MB-COMT over S-COMT is now possible and insight of broader relevance, into the function of bitopic enzymes, is provided.

Inhibitory effects of propyl gallate on membrane lipids metabolism and its relation to increasing storability of harvested longan fruit.

Effects of propyl gallate on membrane lipids metabolism and its relation to storability of harvested longan fruits were studied. The results showed that the propyl gallate-treated longans maintained lower activities of pericarp phospholipase D (PLD), lipase and lipoxygenase (LOX) than those in control fruits. Such treatments could maintain higher levels of pericarp unsaturated fatty acids (USFAs), higher pericarp indices of unsaturated fatty acids (IUFA), and higher pericarp ratio of unsaturated fatty acids to saturated fatty acids (U/S) than those in control fruits. Furthermore, propyl gallate also delayed color changes of pericarp in the harvested longans. Therefore, the postharvest treatments of longan fruits with propyl gallate for increasing storability of longan fruits might be explained by a decrease in activities of PLD, lipase and LOX, and an the increased unsaturation of fatty acids, which could delay membrane lipids metabolism and maintain cell membrane characteristics.

Mechanisms of action of lysophospholipid analogues against trypanosomatid parasites.

Lysophospholipid analogues (LPAs) comprise a class of metabolically stable compounds that have been developed as anticancer agents for over two decades, but which have also potent and selective antiparasitic activity, particularly against trypanosomatid parasites such as Leishmania and Trypanosoma cruzi, both in vitro and in vivo. The in vivo activities of LPAs result from direct effects on their target cells and are not dependent on a functional immune system. Because of their chemical nature, LPAs have a potential for interaction with a variety of subcellular structures and biochemical pathways. However, in mammalian cells LPA-induced growth inhibition and programmed cell death is usually associated with a blockade of phosphatidylcholine (PC) biosynthesis at the level of CTP: phosphocholine citidyltransferase, probably through an increase of cellular ceramide levels due to depressed sphingomyelin synthesis. Although in trypanosomatid parasites much less information is available, inhibition of PC biosynthesis by LPA has also been documented but at the level of phosphatidylethanolamine N-methyl-transferase, as well as LPA-induced classical apoptotic phenomena. The higher activity of LPAs as inhibitors of PC biosynthesis in parasites than in mammalian cells, probably due to different biochemical pathways involved in the two types of cells, could explain their selective antiparasitic action in vivo.

Identification of lipid-phosphatidylserine (PS) as the target of unbiasedly selected cancer specific peptide-peptoid hybrid PPS1.

Phosphatidylserine (PS) is an anionic phospholipid maintained on the inner-leaflet of the cell membrane and is externalized in malignant cells. We previously launched a careful unbiased selection targeting biomolecules (e.g. protein, lipid or carbohydrate) distinct to cancer cells by exploiting HCC4017 lung cancer and HBEC30KT normal epithelial cells derived from the same patient, identifying HCC4017 specific peptide-peptoid hybrid PPS1. In this current study, we identified PS as the target of PPS1. We validated direct PPS1 binding to PS using ELISA-like assays, lipid dot blot and liposome based binding assays. In addition, PPS1 recognized other negatively charged and cancer specific lipids such as phosphatidic acid, phosphatidylinositol and phosphatidylglycerol. PPS1 did not bind to neutral lipids such as phosphatidylethanolamine found in cancer and phosphatidylcholine and sphingomyelin found in normal cells. Further we found that the dimeric version of PPS1 (PPS1D1) displayed strong cytotoxicity towards lung cancer cell lines that externalize PS, but not normal cells. PPS1D1 showed potent single agent anti-tumor activity and enhanced the efficacy of docetaxel in mice bearing H460 lung cancer xenografts. Since PS and anionic phospholipid externalization is common across many cancer types, PPS1 may be an alternative to overcome limitations of protein targeted agents.

Targeting cell membrane adaptation as a novel antimicrobial strategy.

Emergence of antibiotic resistance is an example of the incredible plasticity of bacteria to survive in all environments. The search for new antibiotics active against traditional targets is more challenging due not only to the lack of novel natural products to fulfill the current clinical needs against multidrug-resistant (MDR) bacteria, but also for the possible 'collateral' effects on the human microbiota. Thus, non-traditional approaches to combat MDR bacteria have been proposed. Here, we discuss the possibility of targeting the membrane response to the antibiotic attack (cell membrane adaptation) as a viable strategy to increase the activity of current antimicrobials, enhance the activity of the innate immune system and prevent development of resistance during therapy using the three-component regulatory system LiaFSR of enterococci as a model.

Cobaltous ion inhibition of lipid peroxidation in biological membranes.

The effect of cobalt on lipid peroxidation in biological membranes, phospholipid liposomes and fatty acid micelles was investigated. Cobaltous ion, at micromolar concentrations, inhibited iron-ascorbate induced lipid peroxidation in erythrocyte ghosts, microsomes and phosphatidylserine liposomes at pH 7.4. The pH seemed to be important for the anti-peroxidative effect of cobalt, because under slightly acidic conditions cobalt did not inhibit peroxidation. Cobalt was less effective in inhibiting peroxidation stimulated by organic hydroperoxides. Iron-ascorbate induced lipid peroxidation was also inhibited by EDTA. However, certain ratios of EDTA: cobalt in the reaction mixture stimulated peroxidation. Cobalt did not inhibit lipid peroxidation in linoleic acid micelles and phosphatidylethanolamine liposomes. The presence of phosphatidylserine, however, rendered these micelles and liposomes to cobalt inhibition. We conclude that the cobaltous ion is a potent inhibitor of lipid peroxidation in biological membranes and that the binding of cobalt to phosphatidylserine is necessary for the inhibitory effect of this metal ion.

Membrane disruption and cytotoxicity of hydrophobic N-alkylated imino sugars is independent of the inhibition of protein and lipid glycosylation.

The N-alkyl moiety of N-alkylated imino sugars is crucial for therapeutic activities of these compounds as inhibitors of glycosphingolipid (GSL) biosynthesis and as antivirals. The improved potency afforded by a long N-alkyl moiety is coincident with increased compound-induced cytotoxicity. Therefore, in the present study, we examined the mechanism of this cytotoxicity in detail. Despite N-butyl-deoxynojirimycin and N-butyl-deoxygalactonojirimycin inhibiting the glycosylation of ceramide to glucosylceramide, ceramide levels did not increase in HL60 cells treated with these compounds. Long-chain N-alkylated imino sugars were toxic to cells at concentrations considerably lower than the critical micellar concentrations for these compounds and consequently did not solubilize radioactively labelled cellular proteins and lipids. However, membrane disruption and cell fragmentation did increase in a concentration- and chain-length-dependent manner. These results are consistent with previously proposed interactions between surface-active amphiphiles and protein-containing lipid membranes when drug concentrations are below the critical micellar concentration. Taken together, these results demonstrate that the cellular toxicity of hydrophobic N-alkylated imino sugars is due to cell lysis and cell fragmentation and, most importantly, is not related to the beneficial therapeutic effects of these compounds on protein and in lipid glycosylation. This information will aid in the future development of more selective imino sugar therapeutics for the treatment of human disease.

Drug-induced and postnatal hypothyroidism impairs the accumulation of diacylglycerol in liver and liver cell plasma membranes.

BACKGROUND: Thyroid hormones are well known modulators of signal transduction. The effect of hyper- and hypo-thyroidism on diacylglycerol/protein kinase C (DAG/PKC) signaling in cardiomiocytes has been determined. Triiodothyronine (T3) has been shown to prevent the alpha1-adrenoreceptor-mediated activation of PKC but does not alter the stimulation of enzyme and hepatic metabolism by phorbol ethers. It has been suggested that the elevation of endogenous DAG in senescent or hypothyroid cells changes the PKC-dependent response of cells to phorbol esters and hormones. In the present study, was examined the formation of DAG and activation of PKC in liver cells from rats of different thyroid status. RESULTS: The results obtained provide the first demonstration of DAG accumulation in liver and cell plasma membranes at age- and drug-dependent thyroid gland malfunction. The experiments were performed in either the [14C]CH3COOH-labeled rat liver, liver slices or hepatocytes labeled by [14C] oleic acid and [3H]arachidonic acid or [14C]palmitic acid as well as in the isolated liver cell plasma membranes of 90- and 720-day-old rats of different thyroid status. The decrease of T4 and T3 levels in blood serum of 720-day-old rats and mercazolil-treated animals was associated with increases of both the DAG mass in liver and liver cell plasma membranes and newly synthesized [14C]DAG level in liver and isolated hepatocytes. Hypothyroidism decreased PKC activity in both membrane and cytosol as well as phospholipid and triacylglycerol synthesis in liver. These hypothyroidism effects were restored in liver by injection of T4. T4 administration to the intact animals of different ages decreased the DAG level in liver and isolated plasma membranes and the content of newly synthesized DAG in liver. The reduction of DAG level in liver was not associated with increasing free fatty acid level. DAG labeling ratio 14C/3H in liver slices of rats of different thyroid state sharply differed from PL. DAG was relatively enriched in [14C]oleic acid whereas PL were enriched in [3H]arachidonic acid. CONCLUSIONS: The above data have indicated that thyroid hormones are important physiological modulators of DAG level in rat liver and cell plasma membranes. Age- and drug-induced malfunction of thyroid gland resulted in a prominent decrease of glycerolipid synthesis which may promote DAG accumulation in liver.

Intercellular communication, tumor promotion and non-genotoxic carcinogenesis: relationships based upon structural considerations.

An SAR model for inhibition of metabolic cooperation (iMC) was developed. The structural and physicochemical features associated with the ability to cause iMC are primarily lipophilic moieties consistent with the possibility that they represent receptor-binding ligands. There are also significant parallels between the structural descriptors associated with iMC and those associated with tumor promotion and with carcinogenesis in rodents. Overall, the present study provides structural evidence that iMC is a feature associated with the carcinogenic process.

Hydrostatic pressure decreases membrane fluidity and lipid desaturase expression in chondrocyte progenitor cells.

Membrane biomechanical properties are critical in modulating nutrient and metabolite exchange as well as signal transduction. Biological membranes are predominantly composed of lipids, cholesterol and proteins, and their fluidity is tightly regulated by cholesterol and lipid desaturases. To determine whether such membrane fluidity regulation occurred in mammalian cells under pressure, we investigated the effects of pressure on membrane lipid order of mouse chondrogenic ATDC5 cells and desaturase gene expression. Hydrostatic pressure linearly increased membrane lipid packing and simultaneously repressed lipid desaturase gene expression. We also showed that cholesterol mimicked and cholesterol depletion reversed those effects, suggesting that desaturase gene expression was controlled by the membrane physical state itself. This study demonstrates a new effect of hydrostatic pressure on mammalian cells and may help to identify the molecular mechanisms involved in hydrostatic pressure sensing in chondrocytes.