Interaction of the antifungal ketoconazole and its diphenylphosphine derivatives with lipid bilayers: Insights into their antifungal action.

Ketoconazole (Ke) is an important antifungal drug, and two of its diphenylphosphinemethyl derivatives (KeP: Ph2PCH2-Ke and KeOP: Ph2P(O)CH2-Ke) have shown improved antifungal activity, namely against a yeast strain lacking ergosterol, suggesting alternative modes of action for azole compounds. In this context, the interactions of these compounds with a model of the cell membrane were investigated, using POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) large unilamellar vesicles and taking advantage of the intrinsic fluorescence of Ke, KeP and KeOP. Steady-state fluorescence spectra and anisotropy, including partition and aggregation studies, as well as fluorescence lifetime measurements, were carried out. In addition, the ability of the compounds to increase membrane permeability was assessed through carboxyfluorescein leakage. The membrane/water mole fraction partition coefficients (Kp,x): (3.31 ± 0.36) x105, (8.31 ± 1.60) x105 and (4.66 ± 0.72) x106, for Ke, KeP and KeOP, respectively, show that all three compounds have moderate to high affinity for the lipid bilayer. Moreover, KeP, and particularly KeOP interact more efficiently with POPC bilayers than Ke, which correlates well with their in vitro antifungal activity. Furthermore, although the three compounds disturb the lipid bilayer, KeOP is the quickest and most efficient one. Hence, the higher affinity and ability to permeabilize the membrane of KeOP when compared to that of KeP, despite the higher lipophilicity of the latter, points to an important role of Ph2P(O)CH2- oxygen. Overall, this work suggests that membrane interactions are important for the antifungal activity of these azoles and should be considered in the design of new therapeutic agents.

Effect of Cisplatin and Its Cationic Analogues in the Phase Behavior and Permeability of Model Lipid Bilayers.

Increasing evidence suggests a critical role of lipids in both the mechanisms of toxicity and resistance of cells to platinum(II) complexes. In particular, cisplatin and other analogues were reported to interact with lipids and transiently promote lipid phase changes both in the bulk membranes and in specific membrane domains. However, these processes are complex and not fully understood. In this work, cisplatin and its cationic species formed at pH 7.4 in low chloride concentrations were tested for their ability to induce phase changes in model membranes with different lipid compositions. Fluorescent probes that partition to different lipid phases were used to report on the fluidity of the membrane, and a leakage assay was performed to evaluate the effect of cisplatin in the permeability of these vesicles. The results showed that platinum(II) complex effects on membrane fluidity depend on membrane lipid composition and properties, promoting a stronger decrease in the fluidity of membranes containing gel phase. Moreover, at high concentration, these complexes were prone to alter the permeability of lipid membranes without inducing their collapse or aggregation.

Evaluation of EGCG Loading Capacity in DMPC Membranes.

Catechins are molecules with potential use in different pathologies such as diabetes and cancer, but their pharmaceutical applications are often hindered by their instability in the bloodstream. This issue can be circumvented using liposomes as their nanocarriers for in vivo delivery. In this work, we studied the molecular details of (-)-epigallocatechin-3-gallate (EGCG) interacting with 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) monolayer/bilayer systems to understand the catechin loading ability and liposome stability, using experimental and computational techniques. The molecular dynamics simulations show the EGCG molecules deep inside the lipid bilayer, positioned below the lipid ester groups, generating a concentration-dependent lipid condensation. This effect was also inferred from the surface pressure isotherms of DMPC monolayers. In the polarization-modulated infrared reflection absorption spectra assays, the predominant effect at higher concentrations of EGCG (e.g., 20 mol %) was an increase in lipid tail disorder. The steady-state fluorescence data confirmed this disordered state, indicating that the catechin-induced liposome aggregation outweighs the condensation effects. Therefore, by adding more than 10 mol % EGCG to the liposomes, a destabilization of the vesicles occurs with the ensuing release of entrapped catechins. The loading capacity for DMPC seems to be limited by its disordered lipid arrangements, typical of a fluid phase. To further increase the clinical usefulness of liposomes, lipid bilayers with more stable and organized assemblies should be employed to avoid aggregation at large concentrations of catechin.

Reorganization of plasma membrane lipid domains during conidial germination.

Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted. Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30°C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth.

Formation and Properties of Membrane-Ordered Domains by Phytoceramide: Role of Sphingoid Base Hydroxylation.

Phytoceramide is the backbone of major sphingolipids in fungi and plants and is essential in several tissues of animal organisms, such as human skin. Its sphingoid base, phytosphingosine, differs from that usually found in mammals by the addition of a hydroxyl group to the 4-ene, which may be a crucial factor for the different properties of membrane microdomains among those organisms and tissues. Recently, sphingolipid hydroxylation in animal cells emerged as a key feature in several physiopathological processes. Hence, the study of the biophysical properties of phytosphingolipids is also relevant in that context since it helps us to understand the effects of sphingolipid hydroxylation. In this work, binary mixtures of N-stearoyl-phytoceramide (PhyCer) with palmitoyloleoylphosphatidylcholine (POPC) were studied. Steady-state and time-resolved fluorescence of membrane probes, X-ray diffraction, atomic force microscopy, and confocal microscopy were employed. As for other saturated ceramides, highly rigid gel domains start to form with just ∼5 mol % PhyCer at 24 °C. However, PhyCer gel-enriched domains in coexistence with POPC-enriched fluid present additional complexity since their properties (maximal order, shape, and thickness) change at specific POPC/PhyCer molar ratios, suggesting the formation of highly stable stoichiometric complexes with their own properties, distinct from both POPC and PhyCer. A POPC/PhyCer binary phase diagram, supported by the different experimental approaches employed, is proposed with complexes of 3:1 and 1:2 stoichiometries which are stable at least from ∼15 to ∼55 °C. Thus, it provides mechanisms for the in vivo formation of sphingolipid-enriched gel domains that may account for stable membrane compartments and diffusion barriers in eukaryotic cell membranes.

The role of membrane fatty acid remodeling in the antitumor mechanism of action of 2-hydroxyoleic acid.

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is a potent antitumor drug that we rationally designed to regulate the membrane lipid composition and structure. The lipid modifications caused by 2OHOA treatments induce important signaling changes that end up with cell death (Terés et al., 2012 [1]). One of these regulatory effects is restoration of sphingomyelin levels, which are markedly lower in cancer cells compared to normal cells (Barceló-Coblijn et al., 2011 [2]). In this study, we report another important regulatory effect of 2OHOA on cancer cell membrane composition: a large increase in 2OHOA levels, accounting for ~15% of the fatty acids present in membrane phospholipids, in human glioma (SF767 and U118) and lung cancer (A549) cells. Concomitantly, we observed marked reductions in oleic acid levels and inhibition of stearoyl-CoA desaturase. The impact of these changes on the biophysical properties of the lipid bilayer was evaluated in liposomes reconstituted from cancer cell membrane lipid extracts. Thus, 2OHOA increased the packing of ordered domains and decreased the global order of the membrane. The present results further support and extend the knowledge about the mechanism of action for 2OHOA, based on the regulation of the membrane lipid composition and structure and subsequent modulation of membrane protein-associated signaling.

Insights into the mechanisms underlying the antiproliferative potential of a Co(II) coordination compound bearing 1,10-phenanthroline-5,6-dione: DNA and protein interaction studies.

The very high antiproliferative activity of [Co(Cl)(H2O)(phendione)2][BF4] (phendione is 1,10-phenanthroline-5,6-dione) against three human tumor cell lines (half-maximal inhibitory concentration below 1 µM) and its slight selectivity for the colorectal tumor cell line compared with healthy human fibroblasts led us to explore the mechanisms of action underlying this promising antitumor potential. As previously shown by our group, this complex induces cell cycle arrest in S phase and subsequent cell death by apoptosis and it also reduces the expression of proteins typically upregulated in tumors. In the present work, we demonstrate that [Co(Cl)(phendione)2(H2O)][BF4] (1) does not reduce the viability of nontumorigenic breast epithelial cells by more than 85 % at 1 µM, (2) promotes the upregulation of proapoptotic Bax and cell-cycle-related p21, and (3) induces release of lactate dehydrogenase, which is partially reversed by ursodeoxycholic acid. DNA interaction studies were performed to uncover the genotoxicity of the complex and demonstrate that even though it displays K b (± standard error of the mean) of (3.48 ± 0.03) × 10(5) M(-1) and is able to produce double-strand breaks in a concentration-dependent manner, it does not exert any clastogenic effect ex vivo, ruling out DNA as a major cellular target for the complex. Steady-state and time-resolved fluorescence spectroscopy studies are indicative of a strong and specific interaction of the complex with human serum albumin, involving one binding site, at a distance of approximately 1.5 nm for the Trp214 indole side chain with log K b ~4.7, thus suggesting that this complex can be efficiently transported by albumin in the blood plasma.

A biomimetic platform to study the interactions of bioelectroactive molecules with lipid nanodomains.

In this work, we developed a biomimetic platform where the study of membrane associated redox processes and high-resolution imaging of lipid nanodomains can be both performed, based on a new functional gold modification, l-cysteine self-assembled monolayer. This monolayer proved to be ideal for the preparation of defect-free planar supported lipid bilayers (SLBs) where nanodomains with height difference of ∼1.5 nm are clearly resolved by atomic force microscopy. Single and multicomponent lipid compositions were used, leading to the formation of different phases and domains mimicking the lateral organization of cellular membranes, and in all cases stable and continuous bilayers were obtained. These platforms were tested toward the interaction with bioelectroactive molecules, the antioxidant quercetin, and the hormone epinephrine. Despite the weak interaction detected between epinephrine and lipid bilayers, our biomimetic interface was able to sense the redox process of membrane-bound epinephrine, obtain its surface concentration (9.36 × 10(-11) mol/cm(2) for a fluid bilayer), and estimate a mole fraction membrane/water partition coefficient (Kp) from cyclic voltammetric measurements (1.13 × 10(4) for a fluid phase membrane). This Kp could be used to quantitatively describe the minute changes observed in the photophysical properties of epinephrine intrinsic fluorescence upon its interaction with liposome suspensions. Moreover, we showed that the lipid membrane stabilizes epinephrine structure, preventing its oxidation, which occurs in neutral aqueous solution, and that epinephrine partition and mobility in membranes depends on lipid phase, expanding our knowledge on hormone membrane interactions.

Changes in membrane organization upon spontaneous insertion of 2-hydroxylated unsaturated fatty acids in the lipid bilayer.

Recent research regarding 2-hydroxylated fatty acids (2OHFAs) showed clear evidence of their benefits in the treatment of cancer, inflammation, and neurodegenerative disorders such as Alzheimer's disease. Monolayer compressibility isotherms and isothermal titration calorimetry of 2OHFA (C18-C22) in phosphatidylcholine/phosphatidylethanolamine/sphingomyelin/cholesterol (1:1:1:1 mole ratio), a mixture that mimics the composition of mammalian plasma membrane, were performed to assess the membrane binding capacity of 2OHFAs and their natural, nonhydroxylated counterparts. The results show that 2OHFAs are surface-active substances that bind membranes through exothermic, spontaneous processes. The main effects of 2OHFAs are a decrease in lipid order, with a looser packing of the acyl chains, and a decreased dipole potential, regardless of the 2OHFAs' relative affinity for the lipid bilayer. The strongest effects are usually observed for 2-hydroxyarachidonic (C20:4) acid, and the weakest one, for 2-hydroxydocosahexaenoic acid (C22:6). In addition, 2OHFAs cause increased hydration, except in gel-phase membranes, which can be explained by the 2OHFA preference for membrane defects. Concerning the membrane dipole potential, the magnitude of the reduction induced by 2OHFAs was particularly marked in the liquid-ordered (lo) phase (cholesterol/sphingomyelin-rich) membranes, those where order reduction was the smallest, suggesting a disruption of cholesterol-sphingolipid interactions that are responsible for the large dipole potential in those membranes. Moreover, 2OHFA effects were larger than for both lo and ld phases separately in model membranes with liquid disordered (ld)/lo coexistence when both phases were present in significant amounts, possibly because of the facilitating effect of ld/lo domain interfaces. The specific and marked changes induced by 2OHFAs in several membrane properties suggest that the initial interaction with the membrane and subsequent reorganization might constitute an important step in their mechanisms of action.

Sphingomyelin and sphingomyelin synthase (SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid therapy.

The mechanism of action of 2-hydroxyoleic acid (2OHOA), a potent antitumor compound, has not yet been fully elucidated. Here, we show that human cancer cells have markedly lower levels of sphingomyelin (SM) than nontumor (MRC-5) cells. In this context, 2OHOA treatment strongly augments SM mass (4.6-fold), restoring the levels found in MRC-5 cells, while a loss of phosphatidylethanolamine and phosphatidylcholine is observed (57 and 30%, respectively). The increased SM mass was due to a rapid and highly specific activation of SM synthases (SMS). This effect appeared to be specific against cancer cells as it did not affect nontumor MRC-5 cells. Therefore, low SM levels are associated with the tumorigenic transformation that produces cancer cells. SM accumulation occurred at the plasma membrane and caused an increase in membrane global order and lipid raft packing in model membranes. These modifications would account for the observed alteration by 2OHOA in the localization of proteins involved in cell apoptosis (Fas receptor) or differentiation (Ras). Importantly, SMS inhibition by D609 diminished 2OHOA effect on cell cycle. Therefore, we propose that the regulation of SMS activity in tumor cells is a critical upstream event in 2OHOA antitumor mechanism, which also explains its specificity for cancer cells, its potency, and the lack of undesired side effects. Finally, the specific activation of SMS explains the ability of this compound to trigger cell cycle arrest, cell differentiation, and autophagy or apoptosis in cancer cells.

Anticandidal Cu(I) complexes with neocuproine and 1-(4-methoxyphenyl)piperazine based diphenylaminomethylphosphine: Is Cu-diimine moiety a pharmacophore?

The studies on metal complexes as potential antifungals are of growing interest because they may be the answer to increasingly effective defense mechanisms. Herein we present two new copper(I) iodide or thiocyanide complexes with 2,9-dimethyl-1,10-phenanthroline (dmp) and diphenylphosphine derivative of 1-(4-methoxyphenyl)piperazine (4MP): [CuI(dmp)4MP] (1-4MP) and [CuNCS(dmp)4MP] (2-4MP) - their synthesis, as well as structural and spectroscopic characteristics. Interestingly, while 4MP and its oxide derivative (4MOP) show a very low or no activity against all tested Candida albicans strains (MIC50 ≥ 200 µM against CAF2-1 - laboratory control strain, DSY1050 - mutant without transporters Cdr1, Cdr2, Mdr1; isogenic for CAF2-1, and fluconazole resistant clinical isolates), for 1-4MP and 2-4MP MIC50 values were 0.4 µM, independently on the complex and strain tested. Determination of the viability of NHDF-Ad (Normal Adult Human Dermal Fibroblasts) cell line treated with 1-4MP and 2-4MP showed that for both complexes there was only a 20% reduction in the concentration range » to 2 × MIC50 and the 70% at 4 × MIC50. Subsequently, the MLCT based luminescence of the complexes in aqueous media allowed to record the confocal micrographs of 1-4MP in the cells. The results show that it is situated most likely in the vacuoles (C. albicans) or lysosomes (NHDF-Ad).

Evaluation of the Antiproliferative Properties of CpRu Complexes Containing N-Methylated Triazaphosphaadamantane Derivatives.

Piano-stool-{CpRu} complexes containing 1,3,5-triaza-7-phosphaadamantane (PTA), N-methyl-1,3,5-triaza-7-phosphaadamantane (mPTA), and 3,7-dimethyl-1,3,7-triaza-5-phosphabyciclo[3.3.1]nonane (dmoPTA) were evaluated as drugs against breast cancer. The evaluated compounds include two new examples of this family, the complexes [RuCp(DMSO-κS)(HdmoPTA)(PPh3)](CF3SO3)2 (8) and [RuCp(PPh3)2-µ-dmoPTA-1κP-2κ2N,N'-PdCl2](CF3SO3) (11), which have been synthesized and characterized by NMR, IR, and single-crystal X-ray diffraction. The cytotoxic activity of compounds was evaluated against MDA-MB-231 breast cancer cells, and the three most active complexes were further tested against the hormone-dependent MCF-7 breast cancer cell line. Their cell death mechanism and ruthenium uptake were also evaluated, as well as their binding ability to human serum albumin.

Ruthenium(II) polypyridyl complexes with benzothiophene and benzimidazole derivatives: Synthesis, antitumor activity, solution studies and biospeciation.

With the aim to incorporate pharmacophore motifs into the Ru(II)-polypyridyl framework, compounds [Ru(II)(1,10-phenantroline)2(2-(2-pyridyl)benzo[b]thiophene)](CF3SO3)2 (1) and [Ru(II)(1,10-phenantroline)2(2-(2-pyridyl)benzimidazole)](CF3SO3)2 (2) were prepared, characterized and tested for their antitumor potential. The solid-state structure of the compounds was confirmed by single-crystal X-ray diffraction analysis. The solution behavior of both complexes was investigated, namely their solubility, stability, and lipophilicity in physiological mimetic conditions, as well as an eventual uptake by passive diffusion. In vitro anticancer activity of the complexes on ovarian and different colon cancer cells and apoptosis induction by the complexes were studied. A slow transformation process was observed for complex 1 in aqueous solution when exposed to sunlight, while complex 2 undergoes deprotonation (pKa = 7.59). The lipophilicity of this latter complex depends strongly on the pH and ionic strength. In contrast, 1 is rather hydrophilic under various conditions. Complex 1 was highly cytotoxic on Colo-205 human colon (IC50 = 7.87 µM) and A2780 ovarian (IC50 = 2.2 µM) adenocarcinoma cell lines, while 2 displayed moderate anticancer activity (30.9 µM and 18.0 µM, respectively). The complexes induced late apoptosis and necrosis. Only a weak binding of the complexes to human serum albumin, the main transport protein in blood serum, was found. However, a more significant binding to calf thymus DNA was observed in UV-visible titrations and fluorometric dye displacement studies. Detailed analysis of fluorescence lifetime data collected for the latter systems reveals not only the partial intercalation of the complexes, but goes beyond the usual simplified interpretations.

Gel domains in the plasma membrane of Saccharomyces cerevisiae: highly ordered, ergosterol-free, and sphingolipid-enriched lipid rafts.

The plasma membrane of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid and diphenylhexatriene. Diphenylhexatriene anisotropy is a good reporter of global membrane order. The fluorescence lifetimes of trans-parinaric acid are particularly sensitive to the presence and nature of ordered domains, but thus far they have not been measured in yeast cells. A long lifetime typical of the gel phase (>30 ns) was found in wild-type (WT) cells from two different genetic backgrounds, at 24 and 30 °C, providing the first direct evidence for the presence of gel domains in living cells. To understand their nature and location, the study of WT cells was extended to spheroplasts, the isolated plasma membrane, and liposomes from total lipid and plasma membrane lipid extracts (with or without ergosterol extraction by cyclodextrin). It is concluded that the plasma membrane is mostly constituted by ordered domains and that the gel domains found in living cells are predominantly at the plasma membrane and are formed by lipids. To understand their composition, strains with mutations in sphingolipid and ergosterol metabolism and in the glycosylphosphatidylinositol anchor remodeling pathway were also studied. The results strongly indicate that the gel domains are not ergosterol-enriched lipid rafts; they are mainly composed of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins, suggesting an important role in membrane traffic and signaling, and interactions with the cell wall. The abundance of the sphingolipid-enriched gel domains was inversely related to the cellular membrane system global order, suggesting their involvement in the regulation of membrane properties.

Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts.

Ethanol-lipid bilayer interactions have been a recurrent theme in membrane biophysics, due to their contribution to the understanding of membrane structure and dynamics. The main purpose of this study was to assess the interplay between membrane lateral heterogeneity and ethanol effects. This was achieved by in situ atomic force microscopy, following the changes induced by sequential ethanol additions on supported lipid bilayers formed in the absence of alcohol. Binary phospholipid mixtures with a single gel phase, dipalmitoylphosphatidylcholine (DPPC)/cholesterol, gel/fluid phase coexistence DPPC/dioleoylphosphatidylcholine (DOPC), and ternary lipid mixtures containing cholesterol, mimicking lipid rafts (DOPC/DPPC/cholesterol and DOPC/sphingomyelin/cholesterol), i.e., with liquid ordered/liquid disordered (ld/lo) phase separation, were investigated. For all compositions studied, and in two different solid supports, mica and silicon, domain formation or rearrangement accompanied by lipid bilayer thinning and expansion was observed. In the case of gel/fluid coexistence, low ethanol concentrations lead to a marked thinning of the fluid but not of the gel domains. In the case of ld/lo all the bilayer thins simultaneously by a similar extent. In both cases, only the more disordered phase expanded significantly, indicating that ethanol increases the proportion of disordered domains. Water/bilayer interfacial tension variation and freezing point depression, inducing acyl chain disordering (including opening and looping), tilting, and interdigitation, are probably the main cause for the observed changes. The results presented herein demonstrate that ethanol influences the bilayer properties according to membrane lateral organization.

In vitro Evaluation of Isoniazid Derivatives as Potential Agents Against Drug-Resistant Tuberculosis.

The upsurge of multidrug-resistant tuberculosis has toughened the challenge to put an end to this epidemic by 2030. In 2020 the number of deaths attributed to tuberculosis increased as compared to 2019 and newly identified multidrug-resistant tuberculosis cases have been stably close to 3%. Such a context stimulated the search for new and more efficient antitubercular compounds, which culminated in the QSAR-oriented design and synthesis of a series of isoniazid derivatives active against Mycobacterium tuberculosis. From these, some prospective isonicotinoyl hydrazones and isonicotinoyl hydrazides are studied in this work. To evaluate if the chemical derivatizations are generating compounds with a good performance concerning several in vitro assays, their cytotoxicity against human liver HepG2 cells was determined and their ability to bind human serum albumin was thoroughly investigated. For the two new derivatives presented in this study, we also determined their lipophilicity and activity against both the wild type and an isoniazid-resistant strain of Mycobacterium tuberculosis carrying the most prevalent mutation on the katG gene, S315T. All compounds were less cytotoxic than many drugs in clinical use with IC50 values after a 72 h challenge always higher than 25 µM. Additionally, all isoniazid derivatives studied exhibited stronger binding to human serum albumin than isoniazid itself, with dissociation constants in the order of 10-4-10-5 M as opposed to 10-3 M, respectively. This suggests that their transport and half-life in the blood stream are likely improved when compared to the parent compound. Furthermore, our results are a strong indication that the N' = C bond of the hydrazone derivatives of INH tested is essential for their enhanced activity against the mutant strain of M. tuberculosis in comparison to both their reduced counterparts and INH.

Assessing the role of membrane lipids in the action of ruthenium(III) anticancer compounds.

This work addresses the possible role of the cell membrane in the molecular mechanism of action of two salan-type ruthenium complexes that were previously shown to be active against human tumor cells, namely [Ru(III)(L1)(PPh3)Cl] and [Ru(III)(L2)(PPh3)Cl] (where L1 is 6,6'-(1R,2R)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(3-methoxyphenol); and L2 is 2,2'-(1R,2R)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(4-methoxyphenol)). One-component membrane models were first used, a disordered fluid bilayer of dioleoylphosphatodylcholine (DOPC), and an ordered rigid gel bilayer of dipalmitoylphosphatidylcholine. In addition, two quaternary mixtures of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol were used to mimic the lipid composition either of mammalian plasma membrane (1:1:1:1 mol ratio) or of a cancer cell line membrane (36.2:23.6:6.8:33.4 mol ratio). The results show that both salan ligands L1 and L2 bind relatively strongly to DOPC bilayers, but without significantly affecting their structure. The ruthenium complexes have moderate affinity for DOPC. However, their impact on the membranes was notable, leading to a significant increase in the permeability of the lipid vesicles. None of the compounds compromised liposome integrity, as revealed by dynamic light scattering. Fluorescence spectroscopy studies revealed changes in the biophysical properties of all membrane models analyzed in the presence of the two complexes, which promoted an increased fluidity and water penetration into the lipid bilayer in the one-component systems. In the quaternary mixtures, one of the complexes had an analogous effect (increasing water penetration), whereas the other complex reorganized the liquid ordered and liquid disordered domains. Thus, small structural differences in the metal ligands may lead to different outcomes. To better understand the effect of these complexes in cancer cells, the membrane dipole potential was also measured. For both Ru complexes, an increase in the dipole potential was observed for the cancer cell membrane model, while no alteration was detected on the non-cancer plasma membrane model. Our results show that the action of the Ru(III) complexes tested involves changes in the biophysical properties of the plasma membrane, and that it also depends on membrane lipid composition, which is frequently altered in cancer cells when compared to their normal counterparts.

Organization and dynamics of Fas transmembrane domain in raft membranes and modulation by ceramide.

To comprehend the molecular processes that lead to the Fas death receptor clustering in lipid rafts, a 21-mer peptide corresponding to its single transmembrane domain (TMD) was reconstituted into mammalian raft model membranes composed of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol. The peptide membrane lateral organization and dynamics, and its influence on membrane properties, were studied by steady-state and time-resolved fluorescence techniques and by attenuated total reflection Fourier transformed infrared spectroscopy. Our results show that Fas TMD is preferentially localized in liquid-disordered membrane regions and undergoes a strong reorganization as the membrane composition is changed toward the liquid-ordered phase. This results from the strong hydrophobic mismatch between the length of the peptide hydrophobic stretch and the hydrophobic thickness of liquid-ordered membranes. The stability of nonclustered Fas TMD in liquid-disordered domains suggests that its sequence may have a protective function against nonligand-induced Fas clustering in lipid rafts. It has been reported that ceramide induces Fas oligomerization in lipid rafts. Here, it is shown that neither Fas TMD membrane organization nor its conformation is affected by ceramide. These results are discussed within the framework of Fas membrane signaling events.

C-Glucosylation as a tool for the prevention of PAINS-induced membrane dipole potential alterations.

The concept of Pan-Assay Interference Compounds (PAINS) is regarded as a threat to the recognition of the broad bioactivity of natural products. Based on the established relationship between altered membrane dipole potential and transmembrane protein conformation and function, we investigate here polyphenols' ability to induce changes in cell membrane dipole potential. Ultimately, we are interested in finding a tool to prevent polyphenol PAINS-type behavior and produce compounds less prone to untargeted and promiscuous interactions with the cell membrane. Di-8-ANEPPS fluorescence ratiometric measurements suggest that planar lipophilic polyphenols-phloretin, genistein and resveratrol-act by decreasing membrane dipole potential, especially in cholesterol-rich domains such as lipid rafts, which play a role in important cellular processes. These results provide a mechanism for their labelling as PAINS through their ability to disrupt cell membrane homeostasis. Aiming to explore the role of C-glucosylation in PAINS membrane-interfering behavior, we disclose herein the first synthesis of 4-glucosylresveratrol, starting from 5-hydroxymethylbenzene-1,3-diol, via C-glucosylation, oxidation and Horner-Wadsworth-Emmons olefination, and resynthesize phloretin and genistein C-glucosides. We show that C-glucosylation generates compounds which are no longer able to modify membrane dipole potential. Therefore, it can be devised as a strategy to generate bioactive natural product derivatives that no longer act as membrane dipole potential modifiers. Our results offer a new technology towards rescuing bioactive polyphenols from their PAINS danger label through C-C ligation of sugars.

Biophysical Analysis of Lipid Domains by Fluorescence Microscopy.

The study of the structure and dynamics of membrane domains in vivo is a challenging task. However, major advances could be achieved through the application of microscopic and spectroscopic techniques coupled with the use of model membranes, where the relations between lipid composition and the type, amount and properties of the domains present can be quantitatively studied.This chapter provides protocols to study membrane organization and visualize membrane domains by fluorescence microscopy both in artificial membrane and living cell models of Gaucher Disease (GD ). We describe a bottom-up multiprobe methodology, which enables understanding how the specific lipid interactions established by glucosylceramide, the lipid that accumulates in GD , affect the biophysical properties of model and cell membranes, focusing on its ability to influence the formation, properties and organization of lipid raft domains. In this context, we address the preparation of (1) raft-mimicking giant unilamellar vesicles labeled with a combination of fluorophores that allow for the visualization and comprehensive characterization of those membrane domains and (2) human fibroblasts exhibiting GD phenotype to assess the biophysical properties of biological membrane in living cells using fluorescence microscopy.