Phospholipid Analogues Research Articles

Boronic Acid Based Inhibitors of Autotaxin: Understanding their Biological Role in Terms of Quantitative Structure Activity Relationships (QSAR)

Autotaxin (ATX or NPP2) is a newly discovered secreted glycoprotein lyso-phospholipase D (lysoPLD). Its main role is the lysoPLD activity, which transforms lyso-phosphatidylcholine (LPC) into lyso-phosphatidic acid (LPA). ATX contributes to tumor progression, inflammation, obesity and diabetes and constitutes a target for drug design. Various synthetic phospholipid analogues have been explored as ATX inhibitors. However, potent and selective non-lipid inhibitors of ATX are currently not available. Some new ATX inhibitors have been subjected to a Quantitative-Structure Activity Relationships (QSAR) analysis. CMR represents the calculated molar refractivity of the molecules and seems to govern the ATX inhibition. Steric factors are obviously important. No role for lipophilicity was found. Electronic parameters are not found to be present. Keywords: Boronic Acid based Autotaxin Inhibitors, Hydrophobicity, Molar Refractivity, QSAR, lyso-phosphatidic, cell proliferation, phospholipase, macromolecular systems

Dimerization and Anion Binding of a Fluorescent Phospholipid Analogue

A diarylacetylene fluorophore featuring spatially separated urea and phosphocholine (PC) groups forms a macrocyclic "head-to-tail" dimer stabilized by NH(urea)···OP(PC) hydrogen bonds. At concentrations above ~2 × 10(-5) M in CH(2)Cl(2), the emission intensity of the dimer is quenched by HCO(3)(-) and H(2)PO(4)(-) but not by Cl(-) and NO(3)(-). Under more dilute conditions, all four anions are bound unselectively with association constants on the order of 10(5) M(-1).

Safety evaluation of MJ33 as a potential in vivo inhibitor of NADPH oxidase (NOX2) activation

Inhibition of NOX2 has been proposed for the treatment of acute lung injury (ALI) by preventing the generation of reactive O2 species (ROS). MJ33, a fluorinated phospholipid analogue, functions as a potential ALI therapeutic agent by inhibiting the phospholipase A2 (PLA2) activity that is required for NOX2 activation (J. Biol. Chem., 286:11696, 2011). Long‐term toxicity studies were performed to evaluate the feasibility for this use of MJ33. MJ33 incorporated into mixed unilamellar liposomes was injected either intra‐tracheally (IT) or intravenously (IV) in single or multiple doses, up to 25μmol for a 20g mouse. Mice treated with MJ33 up to 10μmol did not show any obvious changes in clinical score, general appearance, activity level, hair quality, body temperature or feces. At 5μmol by IT or IV route, weight loss was <10% and mice recovered with a subsequent small weight gain. Hematocrit and lung wet to dry weight ratio were not altered by MJ33. Weight loss was greater with 10μmol (in multiple doses: 4× 2.5μmol) but was not progressive. Thus, the maximal tolerated dose (defined as causing >15% reduction in body weight) was 0.42μmol/g body wt (~2000 times higher than the dose shown to inhibit PLA2 activity). LD50 was 1.3μmol/g body wt. These results indicate a high margin of safety for administration of MJ33 in the intact mouse.Supported by NHLBI R01 HL075587

Membrane Assembly Driven by a Biomimetic Coupling Reaction

One of the major goals of synthetic biology is the development of non-natural cellular systems. In this work, we describe a catalytic biomimetic coupling reaction capable of driving the de novo self-assembly of phospholipid membranes. Our system features a coppercatalyzed azide-alkyne cycloaddition that results in the formation of a triazole-containing phospholipid analogue. Concomitant assembly of membranes occurs spontaneously, not requiring preexisting membranes to house catalysts or precursors. The substitution of efficient synthetic reactions for key biochemical processes may offer a general route toward synthetic biological systems.

Synthesis of new derivatives of inositolcontaining phospholipid dimer analogs as potential inhibitors of virus adsorption

The majority of known agents against human immunodeficiency virus (HIV) are targeted at the virus enzymes protease and integrase but do not prevent HIV entry into host cells. Therefore, it is critical to seek small polyanionic molecules as potential inhibitors of virus adsorption. Inositol-containing phospholipids, which are naturally occurring biologically active compounds that are involved in cell regulation, may be promising lead compounds for antiviral drug design. In the present study, dimer analogs of inositol-containing phospholipids were synthesized using the H-phosphonate method. Carboxymethyl and sulfate derivatives based on them were obtained and proposed as potential virus adsorption inhibitors.

Secretory Phospholipase A2 Activity toward Diverse Substrates

We have studied secretory phospholipase A(2)-IIA (sPLA(2)) activity toward different phospholipid analogues by performing biophysical characterizations and molecular dynamics simulations. The phospholipids were natural substrates, triple alkyl phospholipids, a prodrug anticancer etherlipid, and an inverted ester. The latter were included to study head group-enzyme interactions. Our simulation results show that the lipids are optimally placed into the binding cleft and that water molecules can freely reach the active site through a well-defined pathway; both are indicative that these substrates are efficiently hydrolyzed, which is in good agreement with our experimental data. The phospholipid analogue with three alkyl side chains forms aggregates of different shapes with no well-defined sizes due to its cone-shape structure. Phosphatidylglycerol and phosphatidylcholine head groups interact with specific charged residues, but relatively large fluctuations are observed, suggesting that these interactions are not necessarily important for stabilizing substrate binding to the enzyme.

Merozoite release from Plasmodium falciparum-infected erythrocytes involves the transfer of DiIC16 from infected cell membrane to Maurer’s clefts

Merozoite release from infected erythrocytes is a complex process, which is still not fully understood. Such process was characterised at ultra-structural level in this work by labelling erythrocyte membrane with a fluorescent lipid probe and subsequent photo-conversion into an electron-dense precipitate. A lipophilic DiIC16 probe was inserted into the infected erythrocyte surface and the transport of this phospholipid analogue through the erythrocyte membrane was followed up during 48 h of the asexual erythrocyte cycle. The lipid probe was transferred from infected erythrocyte membranes to Maurer’s clefts during merozoite release, thereby indicating that these membranes remained inside host cells after parasite release. Fluorescent structures were never observed inside infected erythrocytes preceding merozoite exit and merozoites released from infected erythrocyte were not fluorescent. However, specific precipitated material was localised bordering the parasitophorous vacuole membrane and tubovesicular membranes when labelled non-infected erythrocytes were invaded by merozoites. It was revealed that lipids were interchangeable from one membrane to another, passing from infected erythrocyte membrane to Maurer’s clefts inside the erythrocyte ghost, even after merozoite release. Maurer’s clefts became photo-converted following merozoite release, suggesting that these structures were in close contact with infected erythrocyte membrane during merozoite exit and possibly played some role in malarial parasite exit from the host cell.Electronic supplementary materialThe online version of this article (doi:10.1007/s00436-011-2314-7) contains supplementary material, which is available to authorized users.

Organization and Dynamics of Cholesterol Crystalline Domains using EPR Spin-Labeling

EPR spin-labeling methods were used to study the organization and dynamics of cholesterol molecules in membranes formed from Chol/PL (cholesterol/phospholipid) mixtures. Membranes made from phospholipids with different cholesterol solubility thresholds (CST) were investigated. It was confirmed using the EPR discrimination methods that cholesterol crystalline domains (CCDs) were present in all of the membrane suspensions when the mixing ratio exceeded the CST. The behavior of phospholipids was monitored with phospholipid analogue spin labels (n-PCs), and cholesterol with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio exceeding significantly the CST. EPR spectra for n-PC indicated that phospholipids exist in these suspensions in the lipid-bilayer-like structures. Spectral characteristics of n-PCs (spin labels located outside the CCD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CCD forms an integral part of the phospholipid bilayer when formed from Chol/PL mixture up to a mixing ratio of 2 to 3. EPR spectra for CSL and ASL in the CCD and the PCD were very similar indicating that in both domains cholesterol exists in the lipid-bilayer-like structures. In the CCD cholesterol molecules are more ordered and sense more rigid environment than in the PCD. This difference is small and can be compared to that induced in the PCD by the ∼10°C decrease of temperature. Thus, cholesterol molecules are unexpectedly dynamic in the CCD what should enhance their active interaction with the PCD. It is suggested that the EPR spin-labeling approach can discriminate the fraction of cholesterol that forms the CCD within the phospholipid bilayer from the fraction that forms the cholesterol structures outside the bilayer.

RETRACTED ARTICLE: Erucylphosphocholine induces growth inhibition, cell cycle arrest, and apoptosis in human choriocarcinoma cells

A membrane-targeted, lipophilic ether lipid of synthetic phospholipid analog, erucylphosphocholine (ErPC), induces apoptosis in some lines of human tumor cells. We investigated the effect of ErPC in the choriocarcinoma cell line, BeWo. BeWo cells were treated with various concentrations of ErPC, and changes in cell growth, the cell cycle, apoptosis, and related parameters were examined. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that BeWo cells were sensitive to the growth inhibitory effect of ErPC. Cell cycle analysis indicated that exposure to ErPC decreased the proportion of cells in the S phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by Annexin V staining of externalized phosphatidylserine and by the loss of mitochondrial transmembrane potential. This induction occurred in conjunction with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. These results suggest that ErPC may serve as a therapeutic agent for the treatment of choriocarcinoma.

Labeling Membranes with Carbocyanine Dyes (DiIs) as Phospholipid Analogs: FIGURE 1.

Cell membranes can be labeled with cationic lipophilic fluorescent carbocyanine dyes that function as phospholipid analogs. These dyes, such as DiI-C(16)(3) (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), label the plasma membrane and (eventually) label all of the membranes within a living cell. The dyes can be also used with model membranes. Carbocyanine dyes are commercially available in different carbon chain lengths (e.g., C(12), C(14), C(18), and C(22)). Depending on the fatty acid composition of the phospholipids in the membrane studied with respect to chain length and degree of saturation, DiIs with different chain lengths can provide optimal incorporation and accurate reporting of membrane motions. DiI-labeled cells can be studied using fluorescence microscopy or fluorescence recovery after photobleaching (FRAP), as well as other techniques.

Arginine diacyl-glycerolipid conjugates as multifunctional biocompatible surfactants

Amino acid glycerolipid conjugates constitute a novel class of lipoamino acids, which can be considered analogues of partial glycerides and phospholipids. They consist of one or two aliphatic chains and one amino acid polar head, linked together through a glycerol moiety. In this article, a description of diacyl glycerolipid conjugates from acetyl-arginine including preparation, physicochemical properties, membrane interaction and its relation with biological properties are described. In particular, we will give details about the following compounds: 1,2-dilauroyl-rac-glycero-3-O-(Nα-acetyl-l-arginine) (1212RAc), with 12 carbon atoms in the alkyl chains and 1,2-dimiristoyl-rac-glycero-3-O-(Nαacetyl- l-arginine) (1414RAc), with 14 carbon atoms in the alkyl chains.

The immiscible cholesterol bilayer domain exists as an integral part of phospholipid bilayer membranes

Electron paramagnetic resonance (EPR) spin-labeling methods were used to study the organization of cholesterol and phospholipids in membranes formed from Chol/POPS (cholesterol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine) mixtures, with mixing ratios from 0 to 3. It was confirmed using the discrimination by oxygen transport and polar relaxation agent accessibility methods that the immiscible cholesterol bilayer domain (CBD) was present in all of the suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST) in the POPS membrane. The behavior of phospholipid molecules was monitored with phospholipid analogue spin labels (n-PCs), and the behavior of cholesterol was monitored with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio of ~2. Additionally, EPR spectra for n-PC, ASL, and CSL indicated that both phospholipids and cholesterol exist in these suspensions in the lipid-bilayer-like structures. EPR spectral characteristics of n-PCs (spin labels located in the phospholipid cholesterol bilayer, outside the CBD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CBD forms an integral part of the phospholipid bilayer when formed from a Chol/POPS mixture up to a mixing ratio of ~2. Interestingly, CSL in cholesterol alone (without phospholipids) when suspended in buffer does not detect formation of bilayer-like structures. A broad, single-line EPR signal is given, similar to that obtained for the dry film of cholesterol before addition of the buffer. This broad, single-line signal is also observed in suspensions formed for Chol/POPS mixtures (as a background signal) when the Chol/POPS ratio is much greater than 3. It is suggested that the EPR spin-labeling approach can discriminate and characterize the fraction of cholesterol that forms the CBD within the phospholipid bilayer.

Modified phospholipids as anti-inflammatory compounds

Oxidized phospholipids (OxPLs) are abundantly found at sites of inflammation and are considered to play an active role in the modulation of the immune response. Whereas most studies attributed a proinflammatory role to OxPLs, recent studies demonstrate that some products of phospholipid oxidation may in fact exhibit anti-inflammatory properties. This study summarizes the proinflammatory and anti-inflammatory properties of OxPLs and sheds light on the therapeutic potential of OxPL derivatives or analogs for treatment of chronic inflammatory disorders. OxPLs may inhibit activation of several Toll-like receptors and can epigenetically reduce the capacity of dendritic cells to function as mature, fully functional immunostimulatory cells. These data demonstrate that OxPLs can induce anti-inflammatory effects. Moreover, VB-201, an orally available synthetic phospholipid analog of the Lecinoxoid family, was found to attenuate inflammation in various preclinical animal models and is currently employed in a phase II clinical trial in psoriasis. Chemical or biological modifications of phospholipids yield various products, some of which may exhibit anti-inflammatory properties. Identification of such species and generation of more stable/potent anti-inflammatory OxPL variants may represent a novel approach for the treatment of immune-mediated diseases such as psoriasis, atherosclerosis, multiple sclerosis and rheumatoid arthritis.

Phospholipidosis Induced by PPARγ Signaling in Human Bronchial Epithelial (BEAS-2B) Cells Exposed to Amiodarone

Phospholipidosis (PL), a disorder characterized by an accumulation of phospholipids in lysosome-derived multilamellar vesicles owing to abnormal lipid metabolism. Amiodarone (AM), an antiarrhythmic drug, can induce pulmonary PL. First, to evaluate potential mechanisms of phospholipidosis, we found lipid metabolism--related genes by microarray. PPARG, FADS2, and SCD out of these genes were key genes in lipid metabolism and PPAR signaling by AM. The messenger RNA (mRNA) levels of PPARG, FADS2, and SCD were upregulated by AM. The PPARγ antagonist GW9662 was used to investigate the possible involvement of PPARγ as a mediator of AM-induced PL, and FADS2 and SCD small interfering RNAs (siRNAs) were used to examine the involvement of FADS2 and SCD in AM-induced PL. The inhibition of PPARγ by GW9662 significantly attenuated the AM-induced upregulation of SCD and slightly decreased the AM-induced upregulation of FADS2. And the pretreatment of GW9662 significantly decreased the AM-induced uptake of the fluorescent phospholipid analog NBD-PC. The siRNA-mediated gene silencing of FADS2 and SCD also decreased the AM-induced NBD-PC uptake. These results suggest that the activation of the PPARγ signaling pathway, including FADS2 and SCD, may play an important role in AM-induced PL. PPARG, FADS2, and SCD are AM-induced PL-related genes and may serve as potential biomarkers for PL caused by pulmonary toxicity. We also provide evidence for a possible mechanism of PL, the accumulation of phospholipid in the induction of FADS2 and SCD by PPARγ, in AM-induced pulmonary toxicity.

Cholesterol Slows down the Lateral Mobility of an Oxidized Phospholipid in a Supported Lipid Bilayer

We investigated the mobility and phase-partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) in supported lipid bilayers. Compared to the conventional phospholipid dihexadecanoylphosphoethanolamine (DHPE)-Bodipy we found consistently higher diffusion constants. The effect became dramatic when immobile obstacles were inserted into the bilayer, which essentially blocked the diffusion of DHPE-Bodipy but hardly influenced the movements of PGPE-Alexa647. In a supported lipid bilayer made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the differences in probe mobility leveled off with increasing cholesterol content. Using coarse-grained molecular dynamics simulations, we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix, concomitant with a translation in the headgroup position of the oxidized phospholipid: at zero cholesterol content, its headgroup is shifted to the outside of the DOPC headgroup region, whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane.

ChemInform Abstract: Synthesis of Phosphonate Analogues of myo-Inositol Phospholipids.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome

The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fluorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (Lo) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38 degrees C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20 degrees -50 degrees C. Calculated electron density profiles indicated the location of 7-DHC was more loosely defined than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome.

ChemInform Abstract: Syntheses of Arsinolipids: Non-isosteric Analogues of Phospholipids.

Reaction of alkaline benzenearsonous and 2,3-dihydroxypropylarsonous acids with rac-glycidol affords the corresponding arsinic acids, which after reduction with thiophenol are acylated with either fatty-acid chlorides/pyridine or fatty acids/dicyclohexylcarbodiimide/4-dimethylaminopyridine and oxidized with hydrogen peroxide to give the arsinolipids (rac-2,3-diacyloxypropyl)phenylarsinic and bis-(rac-2,3-diacyloxypropyl)arsinic acids. The latter is a non-isosteric analogue of bisphosphatidic acid. Copyright © 2000 John Wiley & Sons, Ltd.

Activation of phospholipase A2 by 1-palmitoyl-2-(9'-oxo-nonanoyl)- sn-glycero-3-phosphocholine in vitro

Oxidative stress leads to drastic modifications of both the biophysical properties of biomembranes and their associated chemistry imparted upon the formation of oxidatively modified lipids. To this end, oxidized phospholipid derivatives bearing an aldehyde function, such as 1-palmitoyl-2-(9'-oxo-nonanoyl)- sn-glycero-3-phosphocholine (PoxnoPC) can covalently react with proteins that come into direct contact. Intriguingly, we observed PoxnoPC in a 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) matrix to shorten and abolish the lag time in the action of phospholipase A2 (PLA2) on this composite substrate, with concomitant augmented decrement in pH, indicating more extensive hydrolysis, which was in keeping with enhanced 90° light scattering. The latter was abolished by the aldehyde scavenger methoxyamine, thus suggesting the involvement of Schiff base. Enhanced hydrolysis of a fluorescent phospholipid analogue was seen for PLA2 preincubated with PoxnoPC. Mixing PLA2 with submicellar (22 µM) PoxnoPC caused a pronounced increase in Thioflavin T fluorescence, in keeping with the formation of amyloid-type fibers, which were seen also by electron microscopy.

Peripheral protein organization and its influence on lipid diffusion in biomimetic membranes.

Protein organization on biomembranes and their dynamics are essential for cellular function. It is not clear, however, how protein binding may influence the assembly of underlying lipids or how the membrane structure leads to functional protein organization. Toward this goal, we investigated the effects of annexin a5 binding to biomimetic membranes using fluorescence imaging and correlation spectroscopy. Annexin a5 (anx a5), a peripheral intracellular protein that plays a membrane remodeling role in addition to other functions, binds specifically and tightly to anionic (e.g., phosphatidylserine)-containing membranes in the presence of calcium ion. Our fluorescence microscopy reveals that annexin likely forms assemblies, along with a more dispersed population, upon binding to anionic biomembranes in the presence of calcium ion, which is reflected in its two-component Brownian motion. To investigate the effects of annexin binding on the underlying lipids, we used specific acyl chain labeled phospholipid analogues, NBD-phosphatidylcholine (NBD-PC) and NBD-phosphatidylserine (NBD-PS). We find that both NBD-labeled lipids cluster under anx a5 assemblies, as compared with when they are found under the dispersed annexin population, and NBD-PS exhibits two-component lateral diffusion under the annexin assemblies. In contrast, NBD-PC diffusion is slower by an order of magnitude under the annexin assemblies in contrast to its diffusion when not localized under anx a5 assemblies. Our results indicate that, upon binding to membranes, the peripheral protein annexin organizes the underlying lipids into domains, which may have functional implications in vivo.