Binding of RuCp complexes with human apo-transferrin: fluorescence spectroscopy and molecular docking methods.

The interaction between human serum transferrin (hTf) and three promising organometallic Ru (II)- (η5-C5H5) derived complexes, that have already shown strong in vitro cytotoxicity towards human cancer cell lines, has been investigated using fluorescence spectroscopic techniques. The results suggested that the formation of Ru-hTf systems involves a dynamic collision. The binding process occurs spontaneously (ΔG < 0), mainly driven by hydrophobic interactions. Additional docking studies show that all complexes bind preferably to a specific hydrophobic pocket in the C2-subdomain as already observed for other metal-cyclopentadienyl (MCp) complexes and are in agreement with the experimental results. With these studies we hope to contribute to the understanding of the mechanism of action of these promising cytotoxic agents, thus providing clues for a more rational design.

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.

Phototriggered release of tetrapeptide AAPV from coumarinyl and pyrenyl cages.

Ala-Ala-Pro-Val (AAPV) is a bioactive tetrapeptide that inhibits human neutrophil elastase, an enzyme involved in skin chronic inflammatory diseases like psoriasis. Caged derivatives of this peptide were prepared by proper N- and C-terminal derivatisation through a carbamate or ester linkage, respectively, with two photoactive moieties, namely 7-methoxycoumarin-2-ylmethyl and pyren-2-ylmethyl groups. These groups were chosen to assess the influence of the photosensitive group and the type of linkage in the controlled photo release of the active molecule. The caged peptides were irradiated at selected wavelengths of irradiation (254, 300, and 350 nm), and the photolytic process was monitored by HPLC-UV. The results established the applicability of the tested photoactive groups for the release of AAPV, especially for the derivative bearing the carbamate-linked pyrenylmethyl group, which displayed the shortest irradiation times for the release at the various wavelengths of irradiation (ca. 4 min at 254 nm, 8 min at 300 nm and 46 min at 350 nm).

Biophysical Analysis of Lipid Domains in Mammalian and Yeast Membranes by Fluorescence Spectroscopy.

The use of steady-state and time-resolved fluorescence spectroscopy to study sterol and sphingolipid-enriched lipid domains as diverse as the ones found in mammalian and fungal membranes is herein described. We first address how to prepare liposomes that mimic raft-containing membranes of mammalian cells and how to use fluorescence spectroscopy to characterize the biophysical properties of these membrane model systems. We further illustrate the application of Förster resonance energy transfer (FRET) to study nanodomain reorganization upon interaction with small bioactive molecules, phenolic acids, an important group of phytochemical compounds. This methodology overcomes the resolution limits of conventional fluorescence microscopy allowing for the identification and characterization of lipid domains at the nanoscale.We continue by showing how to use fluorescence spectroscopy in the biophysical analysis of more complex biological systems, namely the plasma membrane of Saccharomyces cerevisiae yeast cells and the necessary adaptations to the filamentous fungus Neurospora crassa , evaluating the global order of the membrane, sphingolipid-enriched domains rigidity and abundance, and ergosterol-dependent properties.

Sphingolipid-enriched domains in fungi.

Plasma membrane carries out multiple physiological functions that require its dynamic and tightly regulated organization into specialized domains of different size, stability, and lipid/protein composition. Sphingolipids are a group of lipids in which the plasma membrane is particularly enriched, thus being crucial for its structure and function. A specific type of sphingolipid-enriched plasma membrane domains, where ergosterol is depleted and lipids are tightly packed in a rigid gel phase, has recently been found in several fungal species, including yeasts and moulds. After presenting the main biophysical features of gel domains and the experimental method for their detection in the fungal plasma membrane, we review these sphingolipid-enriched gel domains and illustrate their importance to both unicellular and multicellular fungi. First, the biophysical properties of the fungal sphingolipid-enriched domains will be analysed taking into consideration the plasma membrane sphingolipidome. Next, their possible biological roles will be summarized, including their relations with plasma membrane compartments and involvement in stress responses. Moreover, since the plasma membrane is a target for several antifungal compounds, a biophysical connection between sphingolipid-enriched domains and antifungal action will be explored.

Yeast Sphingolipid-Enriched Domains and Membrane Compartments in the Absence of Mannosyldiinositolphosphorylceramide.

The relevance of mannosyldiinositolphosphorylceramide [M(IP)2C] synthesis, the terminal complex sphingolipid class in the yeast Saccharomyces cerevisiae, for the lateral organization of the plasma membrane, and in particular for sphingolipid-enriched gel-like domains, was investigated by fluorescence spectroscopy and microscopy. We also addressed how changing the complex sphingolipid profile in the plasma membrane could influence the membrane compartments (MC) containing either the arginine/ H+ symporter Can1p (MCC) or the proton ATPase Pma1p (MCP). To achieve these goals, wild-type (wt) and ipt1Δ cells, which are unable to synthesize M(IP)2C accumulating mannosylinositolphosphorylceramide (MIPC), were compared. Living cells, isolated plasma membrane and giant unilamellar vesicles reconstituted from plasma membrane lipids were labelled with various fluorescent membrane probes that report the presence and organization of distinct lipid domains, global order, and dielectric properties. Can1p and Pma1p were tagged with GFP and mRFP, respectively, in both yeast strains, to evaluate their lateral organization using confocal fluorescence intensity and fluorescence lifetime imaging. The results show that IPT1 deletion strongly affects the rigidity of gel-like domains but not their relative abundance, whereas no significant alterations could be perceived in ergosterolenriched domains. Moreover, in these cells lacking M(IP)2C, a clear alteration in Pma1p membrane distribution, but no significant changes in Can1p distribution, were observed. Thus, this work reinforces the notion that sphingolipid-enriched domains distinct from ergosterol-enriched regions are present in the S. cerevisiae plasma membrane and suggests that M(IP)2C is important for a proper hydrophobic chain packing of sphingolipids in the gel-like domains of wt cells. Furthermore, our results strongly support the involvement of sphingolipid domains in the formation and stability of the MCP, possibly being enriched in this compartment.

New diphenylphosphane derivatives of ketoconazole are promising antifungal agents.

Four new derivatives of ketoconazole (Ke) were synthesized: diphenylphosphane (KeP), and phosphane chalcogenides: oxide (KeOP), sulphide (KeSP) and selenide (KeSeP). These compounds proved to be promising antifungal compounds towards Saccharomyces cerevisiae and Candida albicans, especially in synergy with fluconazole. Simulations of docking to the cytochrome P450 14α-demethylase (azoles' primary molecular target) proved that the new Ke derivatives are capable of inhibiting this enzyme by binding to the active site. Cytotoxicity towards hACSs (human adipose-derived stromal cells) of the individual compounds was studied and the IC50 values were higher than the MIC50 for C. albicans and S. cerevisiae. KeP and KeOP increased the level of the p21 gene transcript but did not change the level of p53 gene transcript, a major regulator of apoptosis, and decreased the mitochondrial membrane potential. Taken together, the results advocate that the new ketoconazole derivatives have a similar mechanism of action and block the lanosterol 14α-demethylase and thus inhibit the production of ergosterol in C. albicans membranes.

Changes in the Biophysical Properties of the Cell Membrane Are Involved in the Response of Neurospora crassa to Staurosporine.

Neurospora crassa is a non-pathogenic filamentous fungus widely used as a multicellular eukaryotic model. Recently, the biophysical properties of the plasma membrane of N. crassa conidia were thoroughly characterized. They evolve during conidial germination at a speed that depends on culture conditions, suggesting an important association between membrane remodeling and the intense membrane biogenesis that takes place during the germinative process. Staurosporine (STS) is a drug used to induce programmed cell death in various organisms. In N. crassa, STS up-regulates the expression of the ABC transporter ABC-3, which localizes at the plasma membrane and pumps STS out. To understand the role of plasma membrane biophysical properties in the fungal drug response, N. crassa was subjected to STS treatment during early and late conidial development stages. Following 1 h treatment with STS, there is an increase in the abundance of the more ordered, sphingolipid-enriched, domains in the plasma membrane of conidia. This leads to higher fluidity in other membrane regions. The global order of the membrane remains thus practically unchanged. Significant changes in sphingolipid-enriched domains were also observed after 15 min challenge with STS, but they were essentially opposite to those verified for the 1 h treatment, suggesting different types of drug responses. STS effects on membrane properties that are more dependent on ergosterol levels also depend on the developmental stage. There were no alterations on 2 h-grown cells, clearly contrasting to what happens at longer growth times. In this case, the differences were more marked for longer STS treatment, and rationalized considering that the drug prevents the increase in the ergosterol/glycerophospholipid ratio that normally takes place at the late conidial stage/transition to the mycelial stage. This could be perceived as a drug-induced development arrest after 5 h growth, involving ergosterol, and pointing to a role of lipid rafts possibly related with an up-regulated expression of the ABC-3 transporter. Overall, our results suggest the involvement of membrane ordered domains in the response mechanisms to STS in N. crassa.

The extracellular matrix modulates H2O2 degradation and redox signaling in endothelial cells.

The molecular processes that are crucial for cell function, such as proliferation, migration and survival, are regulated by hydrogen peroxide (H2O2). Although environmental cues, such as growth factors, regulate redox signaling, it was still unknown whether the ECM, a component of the cell microenvironment, had a function in this process. Here, we showed that the extracellular matrix (ECM) differently regulated H2O2 consumption by endothelial cells and that this effect was not general for all types of cells. The analysis of biophysical properties of the endothelial cell membrane suggested that this modification in H2O2 consumption rates was not due to altered membrane permeability. Instead, we found that the ECM regulated GPx activity, a known H2O2 scavenger. Finally, we showed that the extent of PTEN oxidation was dependent on the ECM, indicating that the ECM was able to modulate H2O2-dependent protein oxidation. Thus, our results unraveled a new mechanism by which the ECM regulates endothelial cell function by altering redox balance. These results pinpoint the ECM as an important component of redox-signaling.

The key role of coligands in novel ruthenium(II)-cyclopentadienyl bipyridine derivatives: Ranging from non-cytotoxic to highly cytotoxic compounds.

A new family of eight ruthenium(II)-cyclopentadienyl bipyridine derivatives, bearing nitrogen, sulfur, phosphorous and carbonyl sigma bonded coligands, has been synthesized. Compounds bearing nitrogen bonded coligands were found to be unstable in aqueous solution, while the others presented appropriate stabilities for the biologic assays and pursued for determination of IC50 values in ovarian (A2780) and breast (MCF7 and MDAMB231) human cancer cell lines. These studies were also carried out for the [5: HSA] and [6: HSA] adducts (HSA=human serum albumin) and a better performance was found for the first case. Spectroscopic, electrochemical studies by cyclic voltammetry and density functional theory calculations allowed us to get some understanding on the electronic flow directions within the molecules and to find a possible clue concerning the structural features of coligands that can activate bipyridyl ligands toward an increased cytotoxic effect. X-ray structure analysis of compound [Ru(η(5)-C5H5)(bipy)(PPh3)][PF6] (7; bipy=bipyridine) showed crystallization on C2/c space group with two enantiomers of the [Ru(η(5)-C5H5)(bipy)(PPh3)](+) cation complex in the racemic crystal packing.

New water-soluble ruthenium(II) cytotoxic complex: biological activity and cellular distribution.

A novel water soluble organometallic compound, [RuCp(mTPPMSNa)(2,2'-bipy)][CF3SO3] (TM85, where Cp=η(5)-cyclopentadienyl, mTPPMS=diphenylphosphane-benzene-3-sulfonate and 2,2'-bipy=2,2'-bipyridine) is presented herein. Studies of interactions with relevant proteins were performed to understand the behavior and mode of action of this complex in the biological environment. Electrochemical and fluorescence studies showed that TM85 strongly binds to albumin. Studies carried out to study the formation of TM85 which adducts with ubiquitin and cytochrome c were performed by electrospray ionization mass spectrometry (ESI-MS). Antitumor activity was evaluated against a variety of human cancer cell lines, namely A2780, A2780cisR, MCF7, MDAMB231, HT29, PC3 and V79 non-tumorigenic cells and compared with the reference drug cisplatin. TM85 cytotoxic effect was reduced in the presence of endocytosis modulators at low temperatures, suggesting an energy-dependent mechanism consistent with endocytosis. Ultrastructural analysis by transmission electron microscopy (TEM) revealed that TM85 targets the endomembranar system disrupting the Golgi and also affects the mitochondria. Disruption of plasma membrane observed by flow cytometry could lead to cellular damage and cell death. On the whole, the biological activity evaluated herein combined with the water solubility property suggests that complex TM85 could be a promising anticancer agent.

Exploring the effect of the ligand design on the interactions between [Ru(η(5)-C5H5)(PPh3)(N,O)][CF3SO3] complexes and human serum albumin.

Ruthenium complexes hold a great potential in chemotherapy as an alternative to the classical platinum based drugs. The organometallic compounds studied in the present work were previously found to exhibit important anticancer activities. Here we have investigated the binding of three ruthenium compounds, namely [Ru(η(5)-C5H5)(PPh3)(bopy)][CF3SO3] 1, [Ru(η(5)-C5H5)(PPh3)(2-ap)][CF3SO3] 2, and [Ru(η(5)-C5H5)(PPh3)(isoquinpk)][CF3SO3] 3 (bopy=2-benzoylpyridine; 2-ap=2-acetylpyridine; isoquinpk=1-isoquinolinyl phenyl ketone) to fatty acid human serum albumin (HSA) and fatty acid-free human serum albumin (HSA(faf)) at physiological pH7.4. The influence of the substituent groups on the heteroaromatic (N,O) coordinated ligand was also studied by fluorescence spectroscopy to get information about this binding. The Stern-Volmer quenching constants (KSV) were calculated at 293, 298 and 310K, with the corresponding thermodynamic parameters ∆G, ∆H and ∆S as well. The fluorescence quenching method was used to determine the number of binding sites (n) and association constants (Ka) at the same temperatures. The binding site to HSA was confirmed by competitive studies of the ruthenium compounds with warfarin.