CRAC motif peptide of the HIV-1 gp41 protein thins SOPC membranes and interacts with cholesterol

This study uses low-angle (LAXS) and wide-angle (WAXS) X-ray synchrotron scattering, volume measurements and thin layer chromatography to determine the structure and interactions of SOPC, SOPC/cholesterol mixtures, SOPC/peptide and SOPC/cholesterol/peptide mixtures. N-acetyl-LWYIK-amide (LWYIK) represents the naturally-occurring CRAC motif segment in the pretransmembrane region of the gp41 protein of HIV-1, and N-acetyl-IWYIK-amide (IWYIK), an unnatural isomer, is used as a control. Both peptides thin the SOPC bilayer by approximately 3 A, and cause the area/unit cell (peptide+SOPC) to increase by approximately 9 A2 from the area/lipid of SOPC at 30 degrees C (67.0+/-0.9 A2). Model fitting suggests that LWYIK's average position is slightly closer to the bilayer center than IWYIK's, and both peptides are just inside of the phosphate headgroup. Both peptides increase the wide-angle spacing d of SOPC without cholesterol, whereas with 50% cholesterol LWYIK increases d but IWYIK decreases d. TLC shows that LWYIK is more hydrophobic than IWYIK; this difference persists in peptide/SOPC 1:9 mole ratio mixtures. Both peptides counteract the chain ordering effect of cholesterol to roughly the same degree, and both decrease KC, the bending modulus, thus increasing the SOPC membrane fluidity. Both peptides nucleate crystals of cholesterol, but the LWYIK-induced crystals are weaker and dissolve more easily.     

Verteporfin, photofrin II, and merocyanine 540 as PDT photosensitizers against melanoma cells

The efficiency of photodynamic effect (PDE) for Photofrin II (PfII), Verteporfin, and Merocyanine 540 (MC540) was compared against neoplastic cells. Triplet state lifetimes and singlet molecular oxygen quantum yields were correlated with biological effect. PfII triplet lifetime was two times longer than that of Verteporfin, however, its singlet molecular oxygen quantum yield was two times lower in comparison with Verteporfin. High singlet molecular oxygen quantum yield of Verteporfin resulted in high biological efficacy. To achieve 50% mortality of cells four times lower light dose and five times lower concentration of Verteporfin were applied in comparison with PfII. The same level of cell damage was reached using 10 times higher light dose and two times higher concentration of MC540 in comparison with PfII. Our results confirm that singlet molecular oxygen based mechanism, prevalent for Verteporfin and PfII, was highly effective against melanoma cells. Verteporfin can be used at small doses with high cellular damage efficiency.     

CRAC motif peptide of the HIV-1 gp41 protein thins SOPC membranes and interacts with cholesterol

This study uses low-angle (LAXS) and wide-angle (WAXS) X-ray synchrotron scattering, volume measurements and thin layer chromatography to determine the structure and interactions of SOPC, SOPC/cholesterol mixtures, SOPC/peptide and SOPC/cholesterol/peptide mixtures. N-acetyl-LWYIK-amide (LWYIK) represents the naturally-occurring CRAC motif segment in the pretransmembrane region of the gp41 protein of HIV-1, and N-acetyl-IWYIK-amide (IWYIK), an unnatural isomer, is used as a control. Both peptides thin the SOPC bilayer by ∼ 3 Å, and cause the area/unit cell (peptide + SOPC) to increase by ∼ 9 Å² from the area/lipid of SOPC at 30 °C (67.0 ± 0.9 Å²). Model fitting suggests that LWYIK's average position is slightly closer to the bilayer center than IWYIK's, and both peptides are just inside of the phosphate headgroup. Both peptides increase the wide-angle spacing d of SOPC without cholesterol, whereas with 50% cholesterol LWYIK increases d but IWYIK decreases d. TLC shows that LWYIK is more hydrophobic than IWYIK; this difference persists in peptide/SOPC 1:9 mole ratio mixtures. Both peptides counteract the chain ordering effect of cholesterol to roughly the same degree, and both decrease K_C, the bending modulus, thus increasing the SOPC membrane fluidity. Both peptides nucleate crystals of cholesterol, but the LWYIK-induced crystals are weaker and dissolve more easily.     

Variation in hydration forces between neutral phospholipid bilayers: evidence for hydration attraction

It is now generally recognized that hydration forces dominate close interactions of lipid hydrophilic surfaces. The commonality of their characteristics has been reasonably established. However, differences in measured net repulsion, particularly evident when phosphatidylethanolamine (PE) and phosphatidylcholine (PC) bilayers are compared, suggest there exists a variety of behavior wider than expected from earlier models of hydration and fluctuation repulsion balanced by van der Waals attraction. To find a basis for this diverse behavior, we have looked more closely at measured structural parameters, degrees of hydration, and interbilayer repulsive forces for the lamellar phases of the following lipids: 1-palmitoyl-2-oleoyl-PE (POPE), egg PE, transphosphatidylated egg PE (egg PE-T), mono- and dimethylated egg PE-T (MMPE and DMPE), 1-stearoyl-2-oleoyl-PC (SOPC), and mixtures of POPE and SOPC. POPE and SOPC bilayers differ not only in their maximum degrees of hydration but also in the empirical hydration force coefficients and decay lengths that characterize their interaction. When mixed with POPE, SOPC effects sudden and disproportionate increases in hydration. POPE, egg PE, and egg PE-T differ in their degree of hydration, molecular area, and hydration repulsion. A single methylation of egg PE-T almost completely converts its hydration and bilayer repulsive properties to those of egg PC; little progression of hydration is seen with successive methylations. In order to reconcile these observations with the conventional scheme of balancing interbilayer hydration and fluctuation-enhanced repulsion with van der Waals attraction, it is necessary to relinquish the fundamental idea that the decay of hydration forces is a constant determined by the properties of the aqueous medium. Alternatively, one can retain that fundamental idea if one recognizes the possibility that polar group hydration has an attractive component to it. In the latter view, that attractive component originates from interbilayer hydrogen-bonded water bridges between apposing bilayer surfaces, arising from correlation of zwitterionic or other complementary polar groups or from factors that affect polar group solubility. The same Marcelja and Radic formalism that accounts so well for the repulsive component also leads to an estimate of the attractive one. We suggest that the full range of degrees of hydration and of interbilayer spacings observed for different neutral bilayers results in part from variable contributions of the attractive and repulsive hydration components.(ABSTRACT TRUNCATED AT 400 WORDS)     

Elastic deformation and failure of lipid bilayer membranes containing cholesterol.

Giant bilayer vesicles were reconstituted from several lipids and lipid/cholesterol (CHOL) mixtures: stearolyloleoylphosphatidylcholine (SOPC), bovine sphingomyelin (BSM), diarachidonylphosphatidylcholine (DAPC), SOPC/CHOL, BSM/CHOL, DAPC/CHOL, and extracted red blood cell (RBC) lipids with native cholesterol. Single-walled vesicles were manipulated by micropipette suction and several membrane material properties were determined. The properties measured were the elastic area compressibility modulus K, the critical areal strain alpha c, and the tensile strength tau lys, from which the failure energy or membrane toughness Tf was calculated. The elastic area expansion moduli for these lipid and lipid/cholesterol bilayers ranged from 57 dyn/cm for DAPC to 1,734 dyn/cm for BSM/CHOL. The SOPC/CHOL series and RBC lipids had intermediate values. The results indicated that the presence of cholesterol is the single most influential factor in increasing bilayer cohesion, but only for lipids where both chains are saturated, or mono- or diunsaturated. Multiple unsaturation in both lipid chains inhibits the condensing effect of cholesterol in bilayers. The SOPC/CHOL system was studied in more detail. The area expansion modulus showed a nonlinear increase with increasing cholesterol concentration up to a constant plateau, indicating a saturation limit for cholesterol in the bilayer phase of approximately 55 mol% CHOL. The membrane compressibility was modeled by a property-averaging composite theory involving two bilayer components, namely, uncomplexed lipid and a lipid/cholesterol complex of stoichiometry 1/1.22. The area expansion modulus of this molecular composite membrane was evaluated by a combination of the expansion moduli of each component scaled by their area fractions in the bilayer. Bilayer toughness, which is the energy stored in the bilayer at failure, showed a maximum value at approximately 40 mol% CHOL. This breakdown energy was found to be only a fraction of the available thermal energy, implying that many molecules (approximately 50-100) may be involved in forming the defect structure that leads to failure. The area expansion modulus of extracted RBC lipids with native cholesterol was compared with recent measurements of intact RBC membrane compressibility. The natural membrane was also modeled as a simple composite made up to a compressible lipid/cholesterol matrix containing relatively incompressible transmembrane proteins. It appears that the interaction of incompressible proteins with surrounding lipid confers enhanced compressibility on the composite structure.     

Evaluation of hydralazine and procainamide effects on fibroblast membrane fluidity

In this study the membrane fluidity of fibroblasts under different pharmacological treatment was investigated. Two drugs, hydralazine and procainamide, were used to treat the immortalized mouse NIH 3T3 and hamster B14 fibroblasts. Membrane lipid dynamics was measured by fluorescence spectroscopy and electron spin resonance techniques. Two kinds of fluorescent probes (TMA-DPH and 12-(9-anthroyloxy)-stearic acid (12-AS)) and two spin labels (5-doxylstearic acid (5-DS) and 12-doxylstearic acid (12-DS)) were used to monitor fluidity in the upper polar and in the hydrophobic core regions of the lipid bilayer. The drugs influenced the membrane hydrophobic core, of which hydralazine induced fluidization and procainamide increased the rigidity. The membrane fluidity at the surface of the lipid bilayer was not modified by the drugs which indicates that both drugs intercalated mainly into the inner core of the cell membrane.     

In vitro photosensitisation of a murine mammary adenocarcinoma cell line with Verteporfin.

Benzoporphyrin derivative monoacid ring A (BPD-MA) is a second generation hydrophobic photosensitiser for PDT that has been approved for ocular disease treatment. In the present paper we report the results of in vitro studies on the photosensitising activity of Verteporfin (liposomally formulated BPD-MA) using an adenocarcinoma derived cell line. Our findings show a quick and efficient uptake of Verteporfin by LM3 cells, reaching maxima concentrations after 5 hr exposure to 18 microg Verteporfin/ml. Independently on the concentration, plateau levels are attained 5 hr after exposure to Verteporfin. Exposure of the cells to the photosensitiser appears to be safe in the darkness within a broad range of concentrations. The hydroxyl radical scavenger mannitol afforded the highest protection against PDT, while L-tryptophan, a well known and efficient singlet oxygen quencher was not an effective protector at all, showing scavenging activity only when it was supplemented at concentration as high as 10 mM and when 50% of the cells were affected, showing that in addition to singlet oxygen, which is considered the primary cytotoxic agent in PDT, other interconvertible reactive oxygen specie (ROS), in particular HO are also generated. Verteporfin-PDT also induced morphological features typical of apoptotic cells. Results of the present work show that the LM3 adenocarcinoma cell can be effectively sensitised with Verteporfin-PDT.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Binding of the antimicrobial peptide temporin L to liposomes assessed by Trp fluorescence

The structure and membrane topology of the antimicrobial peptide temporin L (FVQWFSKFLGRIL- NH(2)) were studied using liposomes as model bilayers. Circular dichroic spectra revealed temporin L to adopt an alpha-helical conformation when bound to liposomes. Binding of temporin L to liposomes induced significant blue shifts of the emission spectra of the single Trp residue (Trp(4)) and also changed its quantum yield. The observed changes in the characteristics of the Trp(4) fluorescence are in keeping with the insertion of this residue into the hydrophobic region of the liposomal bilayers. Access of the aqueous quencher acrylamide to Trp(4) decreased in the sequence 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC)/cholesterol (X(chol) = 0.1) > SOPC > SOPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG, X(POPG) = 0.1) > SOPC/POPG (X(POPG) = 0.2) approximately SOPC/POPG (X(POPG) = 0.4), where X represents molar fraction of the indicated lipid. Whereas quenching of Trp(4) by brominated phospholipids was significant in SOPC liposomes, the quenching efficiency was enhanced when the vesicles contained POPG. The depth of insertion of Trp(4) into lipid bilayers was calculated by both the parallax method and distribution analysis and revealed this residue to reside at an average distance of d approximately 8.0 +/- 0.5 A from the center of both SOPC and SOPC/POPG bilayers. However, in the presence of cholesterol, d was increased to 9.5 +/- 0.5 A, thus revealing Trp(4) to become accommodated more superficially in the bilayer. The above data suggest the presence of two populations of temporin L in SOPC- and POPG-containing membranes with parallel and perpendicular orientation with respect to the plane of the membrane surface.     

Diacylglycerol-rich domain formation in giant stearoyl-oleoyl phosphatidylcholine vesicles driven by phospholipase C activity

We have studied the effect of phospholipase C from Bacillus cereus and Clostridium perfringens (alpha-toxin) on giant stearoyl-oleoyl phosphatidylcholine (SOPC) vesicles. Enzyme activity leads to a binary mixture of SOPC and the diacylglycerol SOG, which phase separates into a SOPC-rich bilayer phase and a SOG-rich isotropic bulk-like domain embedded within the membrane, as seen directly by phase contrast microscopy. After prolonged enzymatic attack, all bilayer membranes are transformed into an isotropic pure SOG phase as characterized by fluorescence microscopy, differential scanning calorimetry, fluorescence anisotropy measurements, and small angle x-ray scattering. These domains may have biological relevance, serving as storage compartments for hydrophobic molecules and/or catalyzing cellular signaling events at their boundaries. Furthermore, in the early stages of asymmetric enzymatic attack to the external monolayer of giant vesicles, we observe a transient coupling of the second-messenger diacylglycerol to membrane spontaneous curvature, which decreases due to enzyme activity, before domain formation and final vesicle collapse occurs.     

Simple, reliable and fast spectrofluorometric method for determination of plasma Verteporfin (Visudyne) levels during photodynamic therapy for choroidal neovascularization.

Photodynamic therapy with Verteporfin, a potent photosensitizer dye, is a very effective treatment for age related macular degeneration due to choroidal neovascularization. Photodynamic therapy offers the potential for selective tissue injury in part attributable to preferential localization of Verteporfin, administrated by intravenous infusion, to the choroidal neovascularization complex and irradiation of the complex with non-laser thermal light at 690 nm resulting in at least temporary thrombosis and vessel closure. Verteporfin is a benzoporphyrin derivative monoacid ring A formulated as a unilamellar liposome. In the blood Verteporfin is associated with lipoprotein fractions and is rapidly cleared via a receptor-mediated uptake mechanism due the high expression of LDL receptors in neovascular tissues. Verteporfin was undetectable in plasma 24 hr after infusion of the recommended dose: 6 mg/m2 of body surface area. The main side effect is photosensitivity of skin which is usually short-lived (24-48 hr) with a low incidence (2.3%). As skin photosensitivity depends on circulating rather than tissue drug levels, we investigate the possibility of developing a simple, fast and reliable spectrofluorometric method to measure plasma Verteporfin levels. Fluorescence emission spectrum (550-750 nm) of 1:10 saline diluted plasma with lambda exc=430 nm showed a characteristic emission peak at 692 nm, the height being proportional to the Verteporfin levels. The sensitivity is around 100 ng/ml and the pharmacokinetics of Verteporfin has been studied from 0 to 5 hr after infusion in six patients older than 65 years with age-related macular degeneration.     

Topography of the prostaglandin endoperoxide H2 synthase-2 in membranes

The topology of association of the monotopic protein cyclooxygenase-2 (COX-2) with membranes has been examined using EPR spectroscopy of spin-labeled recombinant human COX-2. Twenty-four mutants, each containing a single free cysteine substituted for an amino acid in the COX-2 membrane binding domain were expressed using the baculovirus system and purified, then conjugated with a nitroxide spin label and reconstituted into liposomes. Determining the relative accessibility of the nitroxide-tagged amino acid side chains for the solubilized COX-2 mutants, or COX-2 reconstituted into liposomes to nonpolar (oxygen) and polar (NiEDDA or CrOx) paramagnetic reagents allowed us to map the topology of COX-2 interaction with the lipid bilayer. When spin-labeled COX-2 was reconstituted into liposomes, EPR power saturation curves showed that side chains for all but two of the 24 mutants tested had limited accessibility to both polar and nonpolar paramagnetic relaxation agents, indicating that COX-2 associates primarily with the interfacial membrane region near the glycerol backbone and phospholipid head groups. Two amino acids, Phe(66) and Leu(67), were readily accessible to the non-polar relaxation agent oxygen, and thus likely inserted into the hydrophobic core of the lipid bilayer. However these residues are co-linear with amino acids in the interfacial region, so their extension into the hydrophobic core must be relatively shallow. EPR and structural data suggest that membrane interaction of COX-2 is also aided by partitioning of 4 aromatic amino acids, Phe(59), Phe(66), Tyr(76), and Phe(84) to the interfacial region, and by the electrostatic interactions of two basic amino acids, Arg(62) and Lys(64), with the phospholipid head groups.     

Molecular organization of the non-bilayer phospholipid arrangements that induce an autoimmune disease resembling human lupus in mice

Non-bilayer phospholipid arrangements are three-dimensional structures that can form when anionic phospholipids with an intermediate form of the tubular hexagonal phase II (H(II)), such as phosphatidic acid, phosphatidylserine or cardiolipin, are present in a bilayer of lipids. The drugs chlorpromazine and procainamide, which trigger a lupus-like disease in humans, can induce the formation of non-bilayer phospholipid arrangements, and we have previously shown that liposomes with non-bilayer arrangements induced by these drugs cause an autoimmune disease resembling human lupus in mice. Here we show that liposomes with non-bilayer phospholipid arrangements induced by Mn2? cause a similar disease in mice. We extensively characterize the physical properties and immunological reactivity of liposomes made of the zwitterionic lipid phosphatidylcholine and a H(II)-preferring lipid, in the absence or presence of Mn2?, chlorpromazine or procainamide. We use an hapten inhibition assay to define the epitope recognized by sera of mice with the disease, and by a monoclonal antibody that binds specifically to non-bilayer phospholipid arrangements, and we report that phosphorylcholine and glycerolphosphorylcholine, which form part of the polar region of phosphatidylcholine, are the only haptens that block the binding of the tested antibodies to non-bilayer arrangements. We propose a model in which the negatively charged H(II)-preferring lipids form an inverted micelle by electrostatic interactions with the positive charge of Mn2?, chlorpromazine or procainamide; the inverted micelle is inserted into the bilayer of phosphatidylcholine, whose polar regions are exposed and become targets for antibody production. This model may be relevant in the pathogenesis of human lupus.     

Effects of surfactin on membrane models displaying lipid phase separation

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.     

Inhibition of autophagosome formation by the benzoporphyrin derivative verteporfin

Autophagy enables cells to degrade and recycle cytoplasmic materials both as a housekeeping mechanism and in response to extracellular stress such as nutrient deprivation. Recent studies indicate that autophagy also functions as a protective mechanism in response to several cancer therapy agents, making it a prospective therapeutic target. Few pharmacological inhibitors suitable for testing the therapeutic potential of autophagy inhibition in vivo are known. An automated microscopy assay was used to screen >3,500 drugs and pharmacological agents and identified one drug, verteporfin, as an inhibitor of autophagosome accumulation. Verteporfin is a benzoporphyrin derivative used in photodynamic therapy, but it inhibits autophagy without light activation. Verteporfin did not inhibit LC3/Atg8 processing or membrane recruitment in response to autophagic stimuli, but it inhibited drug- and starvation-induced autophagic degradation and the sequestration of cytoplasmic materials into autophagosomes. Transient exposure to verteporfin in starvation conditions reduced cell viability whereas cells in nutrient-rich medium were unaffected by drug treatment. Analysis of structural analogs indicated that the activity of verteporfin requires the presence of a substituted cyclohexadiene at ring A of the porphyrin core but that it can tolerate a number of large substituents at rings C and D. The existence of an autophagy inhibitor among FDA-approved drugs should facilitate the investigation of the therapeutic potential of autophagy inhibition in vivo.     

Concentration effect of cimetidine with POPC bilayer: a molecular dynamics simulation study

All-atom molecular dynamics simulations have been performed on cimetidine in the presence of a palmitoyloleoylphosphatidylcholine (POPC) bilayer. The free energy profile of a single cimetidine molecule passing across POPC bilayer displays a minimum at the interface of bilayer and water. Ten cimetidine molecules were inserted into POPC bilayer to obtain an 8 mol % drug model, and molecular dynamics results showed that cimetidine molecules reside at the polar region of POPC bilayer with sulphur atoms directing to the hydrophobic region. By comparing the one drug model with 8 mol % drug model, one can see that the central barrier to cross the membrane increases while the free energy in bulk water decreases, indicating that the ability of cimetidine passing across the POPC bilayer weakens at increased concentration. In addition, the free energy minimum shifts closer to the hydrophobic core. Our results indicate that with the increased drug concentration, it is more difficult for cimetidine to enter and pass across POPC bilayer.     

Locations and dynamical perturbations for lipids of cationic forms of procaine, tetracaine, and dibucaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy

Locations and dynamical perturbations for lipids of local anesthetics (procaine . HCl, tetracaine . HCl, and dibucaine . HCl) in sonicated egg yolk phosphatidylcholine (PC) vesicles have been studied by 1H-1H nuclear Overhauser effect (NOE) measurements. It was found that tetracaine and dibucaine bind much strongly to the neutral lipids than does procaine and that their mobilities are lowered to such an extent that spin diffusion is transmitted (i.e., omega 2 tau c2 much greater than 1). The intermolecular NOEs between drugs and PC were more effective in the case of dibucaine than with tetracaine, indicating that dibucaine binds to the lipids more strongly than tetracaine; this order agrees well with that of anesthetic potency. However, it was only tetracaine that gave any appreciable dynamical perturbation to the PC vesicles when they were monitored by the extent of transfer of the negative NOE from alpha-methylene protons to choline methyls, olefinic methines, acyl methylenes and terminal methyl protons. This finding was interpreted as being due to the differences in the locations of these drugs in small unilamellar vesicles: (1) procaine interacts with lipids very weakly at the outer surface of the vesicles; (2) tetracaine binds to the lipids both at the outer and inner halves of the bilayer, inserting its rod-like molecule in a forest of acyl chains of PC; (3) dibucaine binds tightly to the polar head-group of PC, which resides only at the outer half of the bilayer vesicles. It was concluded that the relative order of anesthetic potency within these drugs can be correlated not with the ability to affect membrane fluidity but with the ability to bind to lipids at the polar head-group of the bilayer vesicles.     

Effect of pH on the interaction of botulinum neurotoxins A, B and E with liposomes.

The interaction of botulinum neurotoxin serotypes A, B and E with membranes of different lipid compositions was examined by photolabelling with two photoreactive phosphatidylcholine analogues that monitor the polar region and the hydrophobic core of the lipid bilayer. At neutral pH the neurotoxins interacted both with the polar head groups and with fatty acid chains of phospholipids. At acidic pHs the neurotoxins underwent structural changes characterized by a more extensive interaction with lipids. Both the heavy and light chain subunits of the neurotoxins were involved in the process. The change in the nature and extent of toxin-lipid interaction occurred in the pH range 4-6 and was not influenced by the presence of polysialogangliosides. The present data are in agreement with the idea that botulinum neurotoxins enter into nerve cells from a low pH intracellular compartment.     

Hydration of the dienic lipid dioctadecadienoylphosphatidylcholine in the lamellar phase--an infrared linear dichroism and x-ray study on headgroup orientation, water ordering, and bilayer dimensions.

In the phospholipid 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phosphorylcholine (DODPC) in each of the fatty acid chains, a rigid diene group is inserted in a position near the polar/apolar boundary that is exceptionally sensitive for membrane stability. DODPC transforms upon gradual dehydration from the liquid-crystalline to a metastable gel state, which rearranges into two subgel phases at low and intermediate degrees of hydration. The molecular dimensions of the respective bilayers were determined by means of x-ray diffraction. Infrared linear dichroism of selected vibrations of the phosphate and trimethylammonium groups and of the nu13(OH) band of water adsorbed onto the lipid was used to study the molecular order in the polar part of the bilayers in macroscopically oriented samples. The dense packing of the tilted acyl chains in the subgel causes the in-plane orientation of the phosphatidylcholine headgroups with direct interactions between the phosphate and trimethylammonium groups, and a strong orientation of adsorbed water molecules. In the more disordered gel, the thickness of the polar part of the bilayer increases and the lateral interactions between the lipid headgroups weaken. The higher order in the headgroup region of the subgels correlates with shorter decay lengths of the repulsive forces acting between opposite membrane surfaces. This result can be understood if the work to dehydrate the lipid is determined to a certain degree by the work to break up the lipid-water interactions without compensation by adequate lipid-lipid contacts. Almost similar area compressibility moduli are found in the liquid-crystalline and solid phases. Obviously, the lipid avoids lateral stress by the structural rearrangement.