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The anticancer agent edelfosine exhibits a high affinity for cholesterol and disorganizes liquid ordered membrane structures Alessio Ausili, Pablo Martinez Valera, Alejandro Torrecillas, Victoria Gómez-Murcia, Ana M deGodos, Senena Corbalan-Garcia, Jose Antonio Teruel, and Juan C. Gomez-Fernandez Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01539 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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The anticancer agent edelfosine exhibits a high affinity for cholesterol and disorganizes liquid ordered membrane structures

Alessio Ausili, Pablo Martínez-Valera, Alejandro Torrecillas, Victoria Gómez-Murcia, Ana M. de Godos, Senena Corbalán-García, José A. Teruel and Juan C. Gómez Fernández*

Departamento de Bioquímica y Biología Molecular “A”, Facultad de Veterinaria, Regional Campus of International Excellence “Campus Mare Nostrum”, Universidad de Murcia, Apartado de Correos 4021, E-30080-Murcia, Spain.

* Corresponding author. E-Mail: [email protected] Telephone: +34868884766 Fax: + 34 968364147 Keywords: edelfosine; liquid ordered structures; platelet activating factor; lysophosphatidylcholine; anticancer drug.

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Abstract Edelfosine is an anticancer drug with an asymmetric structure because, being a derivative of glycerol, it possesses two hydrophobic substituents of very different lengths. We showed that edelfosine destabilizes liquid ordered membranes formed by either POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), SM (sphingomyelin) and cholesterol (1:1:1 molar ratio) or SM and cholesterol (2:1 molar ratio). This was observed by differential scanning calorimetry where a phase transition arises from either of these membrane systems after the addition of edelfosine. The alteration of the liquid ordered domains was characterized by using small-angle X-ray diffraction that revealed the formation of gel phases as a consequence of the addition of edelfosine at low temperatures and by wide-angle X-ray diffraction that confirmed changes in the membranes indicating the formation of these gel phases. The raise of a phase transition derived by the edelfosine addition was further confirmed by Fourier-transform infrared spectroscopy. The effect of edelfosine was compared with that of structurally analog lipids: PAF (platelet activating factor) and PAPC (1-palmitoyl-2-acetyl-sn-glycero-3phosphocholine), which also have the capacity of destabilizing liquid ordered domains, although they are less potent than edelfosine for this activity, and LPC (lysophosphatidylcholine) that lacks this capacity. It was concluded that edelfosine may associate to cholesterol favorably competing with sphingomyelin and that this set free sphingomyelin to undergo a phase transition. Finally, the experimental observations can be described by molecular dynamics calculations in terms of intermolecular interaction energies in phospholipid-cholesterol membranes. Higher interaction energies between asymmetric phospholipids and cholesterol than between sphingomyelin and cholesterol were obtained. These results are interesting because biophysically characterize one of the main molecular mechanisms to trigger apoptosis of cancer cells.


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Introduction Synthetic alkyl-lysophospholipids (ALP) are analogs of lysophosphatidylcholine (LPC), having antineoplastic and immunomodulating effects derived from their influence on cell signaling pathways. Their structure is mainly characterized by a long hydrocarbon chain that allows easy integration into the lipid bilayer of cell membranes where these compounds accumulate. In this way, they may interfere with normal lipid metabolism, lipid-dependent transduction mechanisms, and membrane structure and dynamics. These actions may explain their antitumor activity 1-2 and also their activity against parasites 3-4. In the case of the antitumor activity, it is interesting that they act through selective apoptosis of tumor cells but they do not interfere with the normal ones 5-6. The first discovered compound of this family which is being already used as anticancer drug was edelfosine (ET-18-O-CH3, 1-octadecyl-2-O-methyl-sn-glycero-3phosphocholine) (see Fig. 1). This compound attracts a very considerable interest for both researchers and pharmaceutical companies since it is known that it specifically affects cancer cells mainly because is preferentially incorporated by tumor cells 5-6. It has been observed that edelfosine is active against leukemic and solid tumors 7-10.

Figure 1. Molecular structures of edelfosine and of the asymmetric lipids structural analogs used in this work. 3 ACS Paragon Plus Environment

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It has been observed that one of the main ways of action of edelfosine is its capacity to perturb liquid ordered domains present in biological membranes, known as rafts 9, 11. Liquid ordered domains in biomembranes are based on the presence of cholesterol. A liquid ordered domain may be formed in a membrane composed by palmitoyloleoylphosphatidylcholine,/sphingomyelin/cholesterol (1:1:1 molar ratio), in equilibrium with liquid disordered. Sphingolipids such as sphingomyelin (SM) are very important components of the liquid ordered domains. Sphingomyelin has been shown to be able to form hydrogen bonds with other phospholipids present in the membrane 12. It has been proposed that the formation of rafts may be determined by formation of both acyl chain 13 and headgroup interactions between sphingolipids 14 with cholesterol packing into the space left between different sphingolipid molecules. Cholesterol shows a preference to interact with sphingomyelin, with respect to a phosphatidylcholine, because of the geometry of both molecules 15. The possible formation of a hydrogen bond between the 3-OH group of cholesterol and the amide of sphingosine has been also discussed by several authors 15-17 although it has not been possible until now to obtain conclusive evidences at this respect. Nevertheless, the formation of a liquid ordered domain may be also determined by changes in the lipid-water interphase and in the network of interactions between the different molecules, between them and with water 17

. The effect of edelfosine in a membrane formed by POPC/SM/cholesterol was

studied by using several techniques like differential scanning calorimetry (DSC), smallangle X-ray diffraction (SAXD) and deuterium nuclear magnetic resonance spectroscopy (2H-NMR), showing that edelfosine may alter membrane organization,


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affecting the presence of liquid ordered phases. In addition, since it was shown that edelfosine colocalizes in cells with rafts, the conclusion was that edelfosine may modify the structure of these liquid ordered structures in cells 18. The effect of edelfosine and of a number of structural analogs of edelfosine on the biophysical properties of model membranes were studied, including PAF (1-alkyl-2acetyl-sn-glycero -3-phosphocholine), PAPC (1-palmitoyl-2-acetyl-sn-glycero-3phosphocholine) and LPC (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine) and it was found that all of them have similar effects on DMPC and on DEPE 19. In this paper we studied edelfosine and the analogs mentioned above with respect to their capacity to alter the domain organization of membranes presenting liquid ordered structures. We used for this purpose DSC, SAXD, wide-angle X-ray diffraction (WAXD), Fouriertransform infrared spectroscopy (FTIR), phosphorus-31 nuclear magnetic resonance spectroscopy (31P-NMR) and transmission electron microscopy (TEM). Our results showed that edelfosine destabilizes liquid ordered domains formed by either POPC/SM/cholesterol (1:1:1 molar ratio) or SM/cholesterol (2:1 molar ratio) and that edelfosine has a higher ability to do that than PAF, PAPC and LPC. We have also used molecular dynamics techniques to investigate the interaction between the asymmetric phospholipids and sphingomyelin with cholesterol concluding that asymmetric phospholipids establish interactions with cholesterol that have higher energies than with sphingomyelin. Materials and Methods Materials PAF (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was obtained from Sigma-Aldrich (Madrid, Spain). PAPC (1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine), POPC (1-


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palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), SM (bovine brain sphingomyelin), LPC (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine), edelfosine (1-octadecyl-2O-methyl-sn-glycero-3-phosphocholine) and cholesterol were obtained from Avanti Polar Lipids (Alabaster, AL, USA). POPC was also obtained as a kind gift from Lipoid (Steinhausen, Switzerland). All others chemicals and reagents were commercial samples of the highest purity. Experimental Methods DSC was carried out by using a Microcal VP Scanning calorimeter (Microcal, Northampton, MA, USA). Lipids, dissolved in chloroform/methanol (2:1 v/v), were mixed in the desired proportions and the organic solvent evaporated under a current of dry nitrogen gas. The last rests of solvents were eliminated under vacuum, leaving the samples for at least 3 hours. Multilamellar vesicles (MLVs) were formed by using 25 mM Hepes, 100 mM NaCl, 0.2 mM EDTA, pH 7.0 and vortexing the samples. 2 mg of phospholipids resuspended in 1 mL of buffer were used for each measurement. Thermograms were analyzed by using Microcal Origin software, supplied by Microcal. For X-ray diffraction, MLV samples were prepared as described above for DSC and 10 mg of phospholipids were used for each measurement. Samples were centrifuged and the pellets placed in a stainless steel holder with cellophane windows. Every diffractogram was obtained with a 10 minutes exposure. SAXD and WAXD were obtained by using a modified Kratky compact cameras (MBraun-Graz Optical System (Graz, Austria) and using a linear position sensitive detector (PSD, MBraun, Garching, Germany). Nickel-filtered Cu Kα X-rays were generated by a Philips PW3830 X-ray generator (Eindhoven, The Netherlands) operating at 50 kV and 30 mA. Calibration of


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the detector was done by using Ag-stearate (small angle region, d-spacing at 48.8 Å) and lupolen (wide-angle region, d-spacing at 4.12 Å). Samples of SM/cholesterol (2:1 molar ratio) and in the presence of 30 mol% of edelfosine or lysophosphatidylcholine were analyzed by FTIR. A total of 4 mg of the phospholipid plus the appropriate amount of edelfosine or lysophosphatidylcholine were employed. The different lipids were dissolved in chloroform/methanol (2:1 v/v), mixed in the adequate proportions and desiccated by evaporating the organic solvents as described above. Multilamellar liposomes were formed by hydrating the samples with 30 µL of 25 mM Hepes, 100 mM NaCl, 0.2 mM EDTA, pH 7.0 buffer and vortexing vigorously. The samples were directly placed into a thermostatted Graseby Specac 20710 cell (Graseby-Specac Ltd., Orpington, Kent, U.K.) fitted with CaF2 windows and 25 µm Teflon spacers. FTIR spectra were recorded with a Bruker Vector 22 Fourier transform infrared spectrometer using a liquid-nitrogen-cooled MCT detector and a normal Beer-Norton apodization function. The spectrometer was continuously purged with dry air during at least 24 h before the experiments and during data acquisition. Spectra of both buffer and samples were acquired under the same scanning and temperature conditions. FTIR spectra were acquired at different temperatures using an external bath circulator. The actual temperature in the cell was controlled by a thermocouple placed directly over the window. A total of 128 scans were carried out for each spectrum with a nominal resolution of 2 cm-1. A sample shuttle accessory was used to obtain the average background and sample spectra. Spectra were collected using the Opus software from Bruker, bands due to buffer were subtracted and the spectra were processed with Opus. Samples for 31P-NMR spectroscopy were prepared as described for DSC by using a total quantity of 15 mg of phospholipid. Samples were hydrated by dispersing 7 ACS Paragon Plus Environment

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them in 25 mM Hepes, 100 mM NaCl, 0.2 mM EDTA, pH 7.0 buffer and multilamellar vesicles were prepared. The samples were then centrifuged and the pellets dispersed in 300 µl of buffer and transferred to NMR glass tubes. 31P-NMR experiments were carried out on a Bruker Avance 600 instrument (Bruker, Etlingen, Germany) spectrometer operating at 242.9 MHz. All spectra were obtained in the presence of a gated broad band proton decoupling (5 W input power during acquisition time), and accumulated free inductive decays were obtained from up to 8000 scans. A spectral width of 48,536 Hz, a memory of 48,536 data points, a 2 s interpulse time, and a 90° radio frequency pulse (11 µs) were used with inverse gated decoupling 1H. Prior to Fourier transformation, an exponential multiplication was applied, resulting in a 100 Hz line broadening. TEM sample was prepared directly placing onto a Formvar film-coated grid a drop of edelfosine/cholesterol (1:1 molar ratio) vesicle suspension, generated as described above. The sample was dried and a drop of 2 % uranyl acetate was successively added to negatively stain the vesicles. When the solvent was evaporated, the grid was inserted in a Philips Tecnai 12 electron microscope and the sample observed. Simulation details All phospholipids have a choline head group and C16 hydrocarbon chains except edelfosine which was constructed with a C18 hydrocarbon chain. The topologies of SM and cholesterol were adapted to Gromos 53A6 force field from Gromos 43A1 20. The topologies of all the other lipid molecules for Gromos 53A6 force field were obtained from the Automated Topology Builder server 21. Packmol 22 was used to generate the initial configuration of asymmetric bilayer with a leaflet composition of 64


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lipid and 64 cholesterol molecules randomly distributed and hydrated with 2500 water molecules, yielding a 1:1 lipid/cholesterol ratio. The lipid bilayer was aligned such that it laid in the XY plane; i.e., the monolayers normal was parallel to the z-axis. Molecular dynamics calculations were performed using Gromacs v5.0.7 molecular simulation package 23. Water was simulated by the simple point charge (SPC) model. All simulations were performed in the NpT ensemble. Pressure control was carried out using the weak-coupling Berendsen scheme with a time constant for coupling of 0.2 ps, semi-isotropic pressure coupling (isotropic in the x and y directions, but different in the z direction) and a constant pressure of 1 bar and compressibility of 4.5 × 10-5 bar-1. Temperature control was carried out with a V-rescale thermostat with a time constant for coupling of 0.2 ps at 298 K 24. Periodic boundary conditions were applied in all directions. All bonds were constraint using the SETTLE algorithm 25 for water and the LINCS algorithm for all other bonds 26. A leapfrog integration scheme with a time step of 2 fs was used. The particle mesh Ewald method was used to correct for long-range electrostatic interactions, and short-range electrostatic and van der Waals interactions were cut off at 1.0 nm. Energy minimization, using the steepest descent algorithm, was done before molecular dynamics simulations to remove any steric clashes or inappropriate geometries. Then a total of 220 ns molecular dynamic simulations were carried out to allow relaxation and equilibration of the systems. At this point we began the production run using the Parrinello–Rahman barostat 27 with a time constant of 1 ps for another 220 ns. The last 40 ns were collected for all calculations. Short range nonbonded interactions energies were calculated with gmx energy utility of Gromacs after rerun the trajectory with the appropriate energy groups. Intermolecular interactions were calculated by excluding atom interactions in a molecule in the topology file and expressed as energy (kJ/mol) per atom of the energy groups involved. PyMOL 1.8.4.0 28


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was used to carry out a rough inspection of the arrangement simulated bilayer. All computations were carried out on super-computers provided by the Computational Service of the University of Murcia (Spain). Results A phase transition is observed after the addition of asymmetric phospholipids to either POPC/SM/cholesterol or SM/cholesterol When edelfosine was added to the POPC/SM (1:1 molar ratio) membrane (Fig. 2A), two transitions were detected, the one appearing at the lowest temperature showed now a Tc of -2 ºC and the other one was shifted, starting at 24 ºC instead of at 13 ºC as it was the case in the absence of edelfosine. It seems that the asymmetric lipid has the effect of partially demixing SM and POPC (Fig. 2A). A similar although less potent effect was detected when 20 mol% of PAF, PAPC and LPC were added. Although in the three cases the same two transitions were seen, ∆H of the transitions were lower than for the samples to which edelfosine was added, so that the transition appearing at the lowest temperature (rich in POPC) had ∆H of 0.59, 0.64, 0.36 and 0.1 kcal/mol, when edelfosine, PAF, PAPC and LPC were added, respectively. Regarding the transition appearing at the highest temperature (rich in SM) ∆H was 3.79, 2.82, 1.43 and 1.30 kcal/mol after the addition of edelfosine, PAF, PAPC and LPC respectively. The transition observed at higher temperature, which must be rich in SM, started at 20 ºC in the cases of LPC and PAF and at 23 ºC in the case of PAPC, compared with 24 ºC for edelfosine and 14 ºC for POPC/SM. These results point to a higher capacity of edelfosine to induce demixing of POPC and SM than the other asymmetric phospholipids (Fig. 2A).


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Figure 2. DSC heating thermograms: (A) of aqueous dispersions of pure POPC; pure SM; POPC/SM (1:1 molar ratio); POPC/SM (1:1 molar ratio) plus 20 mol% of edelfosine (EDF); POPC/SM (1:1 molar ratio) + 20 mol% PAF; POPC/SM (1:1 molar ratio) + 20 mol% PAPC; POPC/SM (1:1 molar ratio) + 20 mol% LPC. (B) of aqueous dispersions of pure POPC; pure SM; POPC/SM/cholesterol (Chol) (1:1:1 molar ratio); POPC/SM/cholesterol (Chol) (1:1:1 molar ratio) + 20 mol% edelfosine (EDF); POPC/SM/cholesterol (Chol) (1:1:1 molar ratio) + 20 mol% PAF; POPC/SM/cholesterol (Chol) (1:1:1 molar ratio) +20 mol% PAPC; POPC/SM/cholesterol (Chol) (1:1:1 molar ratio) + 20 mol% LPC. The concentrations of the lipid mixtures were normalized to 9.8 mM. The heating rate was 60 °C/h.

A ternary mixture POPC/SM/cholesterol (1:1:1 molar ratio) was used as a model membrane in which a Lo (liquid ordered) domain is present. Fig. 2B shows that this mixture did not show any phase transition, unlike what happened with the Lβ to Lα transitions that can be observed for pure POPC (Tc at -4º C), pure SM (Tc at 32 ºC), and 11 ACS Paragon Plus Environment

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the mixture POPC/SM (1:1 molar ratio). The last mixture showed two transitions, evidencing that these two phospholipids are not fully miscible in the gel phase, one of the transitions occurred at the same temperature than pure POPC, with the onset of the transition (Tc) at -4 ºC and the other is a wide transition starting at 13 ºC and ending at 25 ºC. It seems that the first of these transitions corresponds to a phase very rich in POPC, although with a small amount of SM, because it is wider than that shown by pure POPC and slightly shifted to higher temperatures. On the other hand, the second transition seems to correspond to a mixture of both phospholipids but rich in SM because it appears at intermediate temperatures between those of pure phospholipids but closer to the pure SM transition temperature. The sample with a composition POPC/SM/cholesterol (1:1:1) (Fig. 2B) did not show any transition. Interestingly, the addition of 20 mol% edelfosine to this POPC/SM/cholesterol membrane had the unexpected effect of occasioning the appearance of a phase transition at about 17 ºC. This temperature is intermediate between pure POPC and pure SM and the obvious explanation is that it arose from a SM/POPC mixture whereas edelfosine was interacting with some of the cholesterol and thus altering the liquid ordered domain. It is also of note that neither transitions due to pure POPC or pure SM are visible. This is in contrast with what was observed for the mixture POPC/SM (Fig. 2A) in which a small transition at low temperature, attributed to mainly POPC, but the explanation may be that some cholesterol is associated with POPC and, given that the transition of the POPC rich phase appearing at lower temperatures was reduced in ∆H, little cholesterol was enough to make undetectable this transition. It should be mentioned that it was described that cholesterol will distribute almost equally between gel and liquid-crystal phases 29.


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The same types of DSC measurements were carried out for the other asymmetric phospholipids, analogs of edelfosine. Similar results were obtained when these analogs were added to the POPC/SM/cholesterol with a new transition arising for PAF (15.5 ºC) and PAPC (12.5 ºC) but not for LPC (Fig. 2B). It was remarkable that the ∆H of the new transitions were sensibly different with 2.87, 1.03 and 0.79 kcal/mol for the samples to which 20 mol% of edelfosine, PAF and PAPC were respectively added. These results indicate that edelfosine had a stronger effect on the destabilization of the POPC/SM/cholesterol system and that LPC has very little, if any capacity to displace cholesterol from the POPC/SM/cholesterol mixture. A similar study was carried out using a more simplified system, with a membrane formed by SM and cholesterol (2:1 molar ratio). Again in this case the transition observed for pure SM disappeared in SM/cholesterol (2:1 molar ratio) but a transition reappeared in the presence of 30 mol% of edelfosine (Fig. 3). In the last case a transition was observed, starting at 23 ºC. However, the addition of 30 mol% of LPC did not elicit the appearance of a transition, underlining the different capacity to destabilize the membrane organization of different asymmetric phospholipids, in good agreement with the results reported in Fig. 2B.


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Figure 3. DSC heating thermograms of pure SM, SM/cholesterol (Chol) (2:1 molar ratio); SM/cholesterol (Chol) (2:1 molar ratio) + 30 mol% edelfosine (EDF) and SM/cholesterol (Chol) (2:1 molar ratio) + 30 mol% LPC.

X-ray diffraction: SAXD shows that edelfosine, PAF and PAPC, but not LPC, may induce gel phases in both POPC/SM/cholesterol and SM/cholesterol To characterize the biophysical properties of these membrane systems two measurements based on X-ray diffraction were used, the dispersion of small-angle Xray diffraction (SAXD), which provides information on the structure of the membrane on a molecular level and the dispersion of wide-angle X-ray diffraction (WAXD), which relates the molecular data provided by SAXD to greater scale changes, such as the study of the lipid phase in the lipid bilayer. When phospholipids are organized into multilamellar structures they give rise to reflections with relative spacings at 1:1/2:1/3:1/4:1/5 30. This technique not only defines the macroscopic structure itself but also provides the interlamellar repeat distance in the lamellar phase. The largest first order reflection component corresponds to the interlamellar repeat distance (d value),


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which is composed of the bilayer thickness and the thickness of the water layer between bilayers 31.

Figure 4. Small-angle (A, C, G, E and I) and wide-angle (B, D, F, H and J) Xray diffraction profiles at the indicated temperatures of the mixtures POPC/SM/cholesterol (PSC) (1:1:1 molar ratio) (A and B); POPC/SM/cholesterol (1:1:1 molar ratio) + 20 mol% edelfosine (EDF) (C and D); POPC/SM/cholesterol (1:1:1 molar ratio) + 20 mol% PAF (E and F); POPC/SM/cholesterol (1:1:1 molar ratio) + 20 mol% PAPC (G and H); POPC/SM/cholesterol (1:1:1 molar ratio) + 20 mol% LPC (I and J). 15 ACS Paragon Plus Environment

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Fig. 4A shows the SAXD diffractograms of the system POPC/SM/cholesterol (1:1:1 molar ratio). At 5 ºC the d-spacings were 68.5:34.2:23.2:17.2 Å, result compatible with a bilayer membrane. In addition, the d-spacings obtained at 15 ºC (68.5:34.1:23.2:17.2 Å) and at 45 ºC (66.7:33.4:22.5:16.7 Å) were indicative of the same structure. On the other hand, the diffractogram observed for WAXD (Fig. 4B) at the three temperatures were wide and diffuse reflections that indicate absence of gel phases. Fig. 4C shows that after the addition of 20 mol% of edelfosine to a POPC/SM/cholesterol (1:1:1 molar ratio) membrane the diffractogram obtained by SAXD at 5 ºC showed d-spacings at 73.5:36.8:24.7:18.4 Å related as 1:1/2:1/3:1/4, indicating a bilayer structure. At 15 ºC the pattern was the same. At 45 ºC, however it was slightly different with d-spacings at 72.2:35.9 Å related as 1:1/2, indicating also a membrane bilayer. The results obtained through WAXD (Fig. 4D) showed a Bragg reflection at both 5 ºC and 15 ºC appearing at 4.11 Å and at 4.13 Å respectively, both indicative of Lβ phase. At 45 ºC, however, a broad and diffuse reflection appeared that is indicative of a non-gel phase. Very similar results were obtained when 20 mol% of PAF were added to the POPC/SM/cholesterol (1:1:1 molar ratio) membrane (Fig. 4E), showing reflections in the SAXD diffractogram at 70.9:35.8:24.1:17.8 Å at 5 ºC and at 15 ºC and at 45 ºC 71.5:35.9 Å (membrane bilayer in all the cases). The WAXD diffractograms were as for 20% edelfosine in all the cases. When 20 mol% of PAPC were added to the POPC/SM/cholesterol (1:1:1 molar ratio) membrane SAXD diffractograms (Fig. 4G) showed bilayer structures for all the 16 ACS Paragon Plus Environment

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temperatures studied, 5 ºC (70.3:35.2:23.6:17.7 Å), 15 ºC (70.2:35.0:23.6:17.7 Å) and 45 ºC (71.2:35.6 Å). WAXD for this sample (Fig. 4H) showed at 5 ºC a d-spacing at 4.12 Å characteristic of a Lβ gel phase, whereas at 15 and 45 ºC a wide reflection indicated a non-gel phase. After the addition of 20 mol% LPC SAXD diffractograms (Fig. 4I) showed bilayer structures at the three temperatures studied 5 ºC (74.2:37.1:24.8:18.5 Å), 15 ºC (73.5:36.8:24.4:18.4 Å) and 45 ºC (73.5:36.8 Å). WAXD diffractograms were broad and diffuse reflection indicative of a non-gel phase (Fig. 4J). We also studied the simplified system formed by SM/cholesterol (2:1 molar ratio). This sample showed lamellar membranes at the temperatures studied, with dspacings in SAXD (Fig. 5A) at 5 ºC (67.9:33.8:22.5:17.1 Å), 15 ºC (67.3:33.6:22.5:17.0 Å) and 45 ºC (66.7:33.4:22.4:16.8 Å) that indicated membrane bilayers. Also in the presence of 30 mol% of edelfosine (Fig. 5C) and LPC (Fig. 5E) the spacings indicated bilayer structure at the three temperatures studied. In the case of the addition of 30 mol% of edelfosine the d-spacings followed the ratios that indicate membrane bilayer at 5 ºC (72.2:36.1:24.1:18.0 Å), 15 ºC (72.0:36.0:24.0:18.0 Å) and 45 ºC (70.9:35.3:23.4:17.6 Å). The sample with 30 mol% of LPC was in the same case at the three temperatures: 5 ºC (71.5:35.8:23.9:18.0 Å), 15 ºC (70.9:35.5:23.8:17.9 Å) and 45 ºC (69.7:34.5:23.2:17.4 Å). Very interesting were the results observed when these samples were studied by WAXD, since for the sample SM/cholesterol wide and diffuse reflections were observed at the three temperatures, indicating that the membranes were not in gel state (Fig. 5B). The same was observed for the sample to which 30 mol% of lysophosphatidylcholine was added (Fig. 5F). However, the sample containing 30 mol% of edelfosine showed a reflection at 4.10 Å at both 5 ºC and 15 ºC indicating the existence of gel phases as a consequence of the presence of edelfosine and this 17 ACS Paragon Plus Environment

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reflection disappeared at 45 ºC (Fig. 5D) in agreement with the observation of a phase transition made by DSC as described above.

Figure 5. Small-angle (A, C and E) and wide-angle (B, D and F) X-ray diffraction profiles at the indicated temperatures of the mixtures SM/cholesterol (2:1 molar ratio) (A and B); SM/cholesterol (2:1 molar ratio) + 30 mol% edelfosine (EDF) (C and D); SM/cholesterol (2:1 molar ratio) + 30 mol% LPC (E and F).


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Electronic profiles indicated that edelfosine, PAF and PAPC, but not LPC, may induce gel phases in both POPC/SM/cholesterol and SM/cholesterol In order to analyze in more detail the alterations occasioned in the membranes by the different components added, the electronic profiles were calculated using the program GAP (Fig. 6) and the different structural parameters were calculated (Table 1). In the case of the POPC/SM/cholesterol (1:1:1 molar ratio) the distance between the phosphate groups was slightly decreased from 36.4 to 34.8 Å when the temperature was increased from 5 to 45 ºC as it could be expected since no phase transition is undertaken by this mixture in this temperature range. By the same reason dw was not substantially altered either, going from 20 to 19.9 Å. However, when edelfosine was incorporated into this system (at 20 mol%) it was observed that when increasing the temperature from 5 to 45 ºC there was an increase in dHH from 34.6 Å at 5 ºC to 35.2 Å at 45ºC, correlating with the phase transition that edelfosine is producing and dw was decreased from 26.9 to 25.0 Å. Changes of the same sign were also observed after the addition of PAF and PAPC (Table 1), with increases in dHH and decreases in dw when the temperature was increased from 5 to 45 ºC. However, when LPC (20 mol%) was added the observed changes were similar to those of the POPC/SM/cholesterol system for dHH and no change for dw was observed, which is similar to what was observed for the ternary lipid system with no asymmetrical lipid added. If we focus now on changes at a given temperature occasioned by the addition of the asymmetric lipids it can be observed (Table 1) that at 5 ºC dHH was decreased from 36.4 to 34.6, 32.2 and 31.6 Å after the addition of edelfosine, PAF and PAPC respectively; this is associated to the formation of Lβ phases in these systems. Practically no alteration was however observed in the presence of LPC (36.4 in the absence and 36.2 Å in its presence), again in agreement with the no formation of a gel phase after the addition of LPC. However, 19 ACS Paragon Plus Environment

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minor changes were observed at 45 ºC, when all the systems are fluid as a consequence of the addition of asymmetric lipids. On the other hand, dw suffered also changes that pointed after all the additions of asymmetric lipids and at both temperatures, to an increase probably due to the increase in the presence of the phosphocholine group in the lipid-water interface. Similar results were also obtained for the simplified system with SM/cholesterol (2:1 molar ratio). Again, dHH was decreased for this system from 35.8 to 35.0 Å when the temperature was increased from 5 to 45 ºC and dw suffered very little change from 20.1 to 19.7 Å. When 30 mol% LPC was added dHH decreased from 35.0 to 34.4 Å and dw from 25.2 to 24.5 Å. However, if 30 mol% of edelfosine was added dHH was increased from 34.6 to 36.0 Å and dw decreased from 24.9 to 21.7 Å. As in the ternary lipid mixture, also in this system the addition of edelfosine at 5 ºC occasioned a decrease in dHH from 35.8 to 34.6 Å, whereas LPC also produced a more modest change to 35.0 Å, whereas very little changes were observed at 45 ºC as a consequence of the addition of either edelfosine or LPC. With respect to dw substantial increases were again observed when edelfosine or LPC were added as a consequence of the increase in the phosphocholine group at the lipid-water interface.


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Figure 6. Electronic profiles obtained by using the GAP program from small-angle X-ray diffraction at 5 ºC (A and C) and 45 ºC (B and D). PSC stands for a POPC/SM/cholesterol

mixture (1:1:1 molar ratio); PSC + edelfosine, PSC + PAF, PSC + PAPC and PSC + LPC for PSC membranes containing 20 mol% of edelfosine, 20 mol% of PAF, 20 mol% of PAPC or 20 mol% of LPC, respectively. SM/Chol stands for a SM/cholesterol (2:1 molar ratio) mixture; SM/Chol + edelfosine and SM/Chol + LPC stands for SM/cholesterol (2:1 mixture) with the addition of 30 mol% edelfosine or LPC, respectively.


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Table 1. Fitting parameters: d stands for the lamellar repeat distance. The following parameters were obtained by using the GAP program, as described in Materials and Methods; dHH, headgroup peak-peak distance; dw, water layer. POPC/SM/cholesterol mixture (1:1:1 molar ratio), with the addition of 20 mol% of edelfosine (EDF), of PAF, of PAPC and of LPC. SM/cholesterol (2:1 molar ratio) mixture with the addition of 30 mol% of edelfosine (EDF) and of LPC. POPC/SM/cholesterol POPC/SM/cholesterol POPC/SM/cholesterol/EDF POPC/SM/cholesterol/EDF POPC/SM/cholesterol/PAF POPC/SM/cholesterol /PAF POPC/SM/cholesterol /PAPC POPC/SM/cholesterol /PAF POPC/SM/cholesterol /LPC POPC/SM/cholesterol/LPC SM/cholesterol SM/cholesterol SM/cholesterol/EDF SM/cholesterol/EDF SM/cholesterol/LPC SM/cholesterol/LPC

T (ºC) 5 45 5 45 5 45 5 45 5 45 5 45 5 45 5 45

d (Å) 68.5 66.7 73.5 72.2 70.9 71.5 70.3 71.2 72.8 70.2 67.9 66.7 71.5 69.7 72.2 70.9

dHH (Å) 36.4 34.8 34.6 35.2 32.2 35.8 31.6 34.8 36.2 33.6 35.8 35.0 34.6 36.0 35.0 34.4

dw (Å) 20.0 19.9 26.9 25.0 26.7 23.7 26.7 24.4 24.6 24.6 20.1 19.7 24.9 21.7 25.2 24.5

Infrared spectroscopy confirms that edelfosine induces the appearance of a phase transition An additional and independent technique, Fourier-transform infrared spectroscopy (FTIR), was used to confirm the different actions of edelfosine and LPC on the SM/cholesterol (2:1 molar ratio) (Fig. 7).


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Figure 7. The maximum of the CH2 symmetric stretching band of the acyl chains of the lipids obtained by FTIR spectroscopy. Samples of pure SM, SM/cholesterol (2:1 molar ratio), SM/cholesterol plus 30 mol% of edelfosine (EDF) and SM/cholesterol plus 30 mol% of LPC are shown.

When the temperature is increased, a shift in frequency of the maximum of the CH2 stretching mode which takes place during the endothermic phase transition of SM is observed that can be associated with the change from all-trans to gauche conformers 32

and hence the frequencies of these bands are related to the average number of gauche

conformers. Fig. 7 shows that the phase transition induces a shift in frequency from 2847.4 cm-1 (all trans) to 2849.5 cm-1 (gauche) in pure SM, the onset of the transition being located at 34 ºC in agreement with the DSC measurements. However, the sample SM/cholesterol (2:1 molar ratio) did not show any phase transition, again in agreement with the DSC results. Interestingly the addition of 30 mol% of edelfosine to the SM/cholesterol membrane, but not that of 30 mol% of LPC, elicits a phase transition starting at 24 ºC, again in close agreement with the results observed by DSC (see Fig. 3) and these results are perfectly compatible with those obtained by means of X-ray diffraction. 23 ACS Paragon Plus Environment

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Edelfosine may interact with cholesterol forming bilayer vesicles The ability of edelfosine to strongly interact with cholesterol can be evidenced by its ability to form bilayer vesicles. Fig. 8A shows the 31P-NMR spectra of pure edelfosine and a mixture edelfosine/cholesterol (1:1 molar ratio). It can be seen that, whereas the sample of pure edelfosine showed an isotropic peak, the spectrum of the mixture edelfosine/cholesterol was anisotropic indicating the formation of relatively big vesicles and Fig. 8A shows that two types of particles can be detected. There is an anisotropic pattern that probably corresponds to big unilamellar or even multilamellar vesicles and a superimposed isotropic peak which could arise from either very small vesicles or micelles. Fig. 8B shows that these two different populations can be also detected by using transmission electron microscopy after negative staining of the vesicles, with small vesicles and also some bigger ones. Bilayer structures were also seen in the case of equimolar mixtures of the other three asymmetric phospholipids, namely PAF, PAPC and LPC, with cholesterol. Bilayer structures were seen through 31

P-NMR and vesicles by electron microscopy. It should be stated that pure cholesterol

has a very low solubility in water and it has been described that it can form micelles at nanomolar concentrations but not bilayers 33.


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Figure 8. Part A depicts the 31P-NMR spectra of pure edelfosine and edelfosine/cholesterol (1:1 molar ratio). Part B corresponds to an electron microscopy image of vesicles of edelfosine/cholesterol (1:1 molar ratio) stained with uranyl acetate. The bar corresponds to 1 µm. Molecular Dynamics In order to understand the interaction between the asymmetric phospholipids and cholesterol, comparing these interactions with that between sphingomyelin and cholesterol, we have carried out a molecular dynamics study of edelfosine, PAF, PAPC, LPC and SM with cholesterol. The area per molecule is a measurement of the average lateral area for lipid and cholesterol molecules in a lipid bilayer composed of a mixture of phospholipids and cholesterol. This parameter is often used as an indicator to monitor equilibration during 25 ACS Paragon Plus Environment

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simulations. In this work the area per molecule (AM) has been calculated by using equation 1, where ABOX is the area of the simulation box, NL the number of phospholipid molecules and NCHOL the number of cholesterol molecules.

Eq. 1

The equilibration of the lipid membranes used is demonstrated by the stability of the simulations revealed in the near-flatness, within fluctuations, of the plot of the area per molecule for the last 40 ns simulation time (Fig. 9).

Figure 9. Instant surface area per molecule in the simulated lipid/cholesterol membranes: SM (black), LPC (red), EDF (blue), PAPC (light blue), PAF (green).

The averaged values of AM are shown in Table 2. The area per phospholipid, AL, and the area per cholesterol, ACHOL (Table 2) can be evaluated for these membranes by equations 2 and 3, according to the procedure described by

34

. Where VBOX is the

volume of the simulation box, NTL is the total number of molecules (NTL=NL+NCHOL), r 26 ACS Paragon Plus Environment

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is the cholesterol to phospholipid ratio (r=NCHOL/NL), NW is the number of water molecules in the simulation box, and VW and VCHOL are the volumes of water and cholesterol molecules taken as 0.0321 nm3 and 0.593 nm3, respectively 34.

2 ⋅ ⋅ ⋅ Eq. 2 ⋅ 1 − 1 − ⋅ − ⋅

2 ⋅ ⋅ − ⋅

Eq. 3

Table 2. Surface area of phospholipid/cholesterol (1:1 molar ratio) membranes

AM (nm2) ACHOL (nm2) AL (nm2) SM

0.42

0.28

0.57

LPC

0.29

0.25

0.33

PAPC

0.30

0.25

0.35

PAF

0.30

0.25

0.35

EDF

0.29

0.24

0.35

A higher value of the area per phospholipid is obtained for the SM membrane (Table 2). This is in accordance to the presence of two acyl chains in the SM molecule as compared to the rest of the phospholipids with only one acyl chain, which show a similar area per lipid value for the four asymmetric phospholipids. It is difficult to compare these results with the literature by the lack of experimental and simulated data of similar bilayer membranes in the conditions used in this work. Thus, the reported data are few and varying. For instance, a wide range of values have been reported for the area per lipid value for SM membrane at 50 ºC, ranging from 0.47 nm2

35

to 0.64

nm2 36. For SM-CHOL (2:1) membranes a value of 0.513 nm2 and 0.272 nm2 have been reported by MD simulations at 20 ºC for AL and ACHOL values respectively 37, which is in good agreement with our result. 27 ACS Paragon Plus Environment

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Figure 10. Radial distribution function from lipid to cholesterol in the XY plane: SM (black), LPC (red), EDF (pink), PAPC (green), PAF (blue).

The radial distribution function, g(r), for the lipid-cholesterol membranes was calculated to measure the probability of finding a cholesterol molecule with respect to the distance from a lipid molecule in the surface of the lipid layer, the XY plane (Fig. 10). This measure is useful to estimate the lateral ordering of lipid bilayers. The first peak appears at a most probable distance to find a cholesterol molecule around a lipid molecule. It can be seen that a lower g(r) value is obtained for the SM membrane and at a higher distance indicating that fewer cholesterol molecules are found around SM molecule and at a higher distance than the rest of the phospholipids.


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Figure 11. Total energy of the simulated phospholipid-cholesterol membranes at a 1:1 phospholipid/cholesterol molar ratio.

It has been shown in this work that the addition of asymmetric phospholipids to a SM-cholesterol membrane tends to restore the melting transition of the SM membrane without cholesterol (Fig. 3). Thus, in order to study the possible differential interactions of these phospholipids with cholesterol the energetics of the molecular interactions in these lipid-cholesterol bilayers have been calculated to describe how cholesterol segregation induced by asymmetric phospholipids could take place to allow SM melting. The total energies for intermolecular interactions of the lipid-cholesterol bilayers are shown in Fig. 11. The lowest value of total energy (-14.4 kJ/mol) was found for the edelfosine/cholesterol bilayer, while the highest value was for the SM/cholesterol bilayer (-8.8 kJ/mol), meaning higher intermolecular interaction energy in the edelfosine/cholesterol bilayer than in the SM/cholesterol one. In order to find the origin of these differences the intermolecular interaction energy was decomposed into electrostatic, Ecoul, and Lennard-Jones contributions, ELJ. The first one, Ecoul, accounts for the Coulomb interaction between charged atoms pairs, and the second one, the 29 ACS Paragon Plus Environment

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Lennard-Jones potential, describes the van der Waals interactions which is frequently used in force fields of large molecules because of its simplicity 38.

Figure 12. Intermolecular interaction energies in the simulated lipid-cholesterol membranes decomposed as short range electrostatic (black bars) and van der Waals (grey bars) interactions. In white bars the sum of the two energies are represented. 30 ACS Paragon Plus Environment

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Fig. 12 shows the intermolecular interaction energies of phospholipidphospholipid, phospholipid-cholesterol and cholesterol-cholesterol, corresponding to the electrostatics, van der Waals and total interactions as the sum of them. The main energy contribution to phospholipid-phopholipid interactions corresponds to the electrostatic interactions, being higher for SM and LPC, hence, the same trend is observed for the total phospholipid-phospholipid interaction (Fig. 12A). The phospholipid-cholesterol interaction energies arise mainly from the van der Waals energy term. An increase in the van der Waals and electrostatic interaction energies is found in the asymmetric phospholipids as compared to the SM (Fig. 12B). Fig. 12C shows the cholesterolcholesterol interaction energies. The electrostatic contribution is negligible, the main contribution corresponds to the van der Waals interactions as expected for a molecule with high hydrophobicity. A smaller energy value was obtained for the SM/cholesterol bilayer as compared to the rest of phospholipids. The main differences observed between SM and the asymmetric phospholipids lie in the phospholipid-cholesterol interaction, mainly due to the electrostatic interactions (Fig. 12B). Since the hydrogen bonds contribute to the electrostatic interaction energy, the average number of hydrogen bonds formation in the simulated lipid bilayer, have been calculated. In this work, geometric criteria have been used to find a hydrogen bond (Hbond) from the simulation trajectory: the acceptor-hydrogen distance dah ≤ 0.35 nm and the hydrogen-donor-acceptor angle θhda ≤ 30° used as default option in the Gromacs software package. Table 3 shows the intra- and intermolecular average number of Hbonds between two different phospholipids and between a phospholipid and cholesterol. It is to be noted that only SM and LPC are able to form H-bonds. In the case of SM, most of the H-bonds are intramolecular and only 0.35 H-bonds per SM molecule are


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intermolecular. However, in LPC the number of intramolecular H-bonds is negligible. Intramolecular SM H-bonds are present in virtually every SM molecule, ~1 H-bond/SM (Table 3), and it has been found to be present and very stable in other simulations of SM bilayers

39-41

. However, only intermolecular H-bonds would contribute to the

electrostatic intermolecular interaction energy term. On the other hand, with respect to phospholipid-cholesterol H-bonds, SM is the phospholipid which forms less H-bonds with cholesterol. Similar results have been also reported for SM-cholesterol membranes in similar membrane systems, eg., sphingomyelin (18:0) and cholesterol (60/40) dilute

cholesterol

and

SM

in

a

POPC

matrix

in

molar

42

fractions

or of

POPC/PSM/cholesterol 62:1:1 41.

Table 3. Average number of hydrogen bonds per phospholipid molecule Intramolecular Intermolecular Phospholipid- PhospholipidPhospholipid Phospholipid

PhospholipidCholesterol

0.98

0.35

0.41

0

0

0.63

LPC

0.04

0.13

0.75

PAF

0

0

0.83

EDF

0

0

0.75

SM PAPC

Discussion Edelfosine has been shown to localize in membrane rafts 18, 43 and to be able of disturbing the SM-cholesterol interaction 18, 44-45, as reviewed 46. The results obtained in this study extend our biophysical understanding of how edelfosine is able of acting as a destabilizer agent of systems possessing a liquid ordered structure, provided by the interaction of cholesterol with phospholipids such as POPC


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and SM. This is deduced from the appearance of a phase transition in both POPC/SM/cholesterol (1:1:1 molar ratio) and SM/cholesterol (2:1 molar ratio) when edelfosine was added. This was clearly certified by DSC and FTIR and by the observation of a Lβ phase by means of WAXD. The results obtained by SAXD and analyzed using the GAP program also indicate the existence of a phase transition in the presence of edelfosine in the two membrane systems used as platforms in this work. It is also noteworthing that edelfosine may form bilayer vesicles when associated with cholesterol and this explain why it was observed in a previous report that when combined in vesicles will produce less hemolytic effects, confirming that when dispersed in water, these two lipids form liposomes 47-48. This effect should be due to the complementary forms of edelfosine (inverted cone shape) and cholesterol (cone shape) 47-48 and we propose that this combination could be easily formed in the cell membrane facilitating the removing of cholesterol from liquid ordered phases. The strong interactions of edelfosine with cholesterol have been observed in monolayer studies. It should be stated that the concentration of edelfosine used was indeed higher than what should be expected in cells, but these high concentrations are used in order to be able to visualize the effects when using biophysical techniques. However the 1:1 edelfosine/ cholesterol interaction is expected to occur in cell membranes even at lower concentrations. We have compared the effects of edelfosine with those of other asymmetric phospholipids that contain phosphocholine as polar group, as it is the case of PAF, PAPC and LPC. It is interesting that PAF and PAPC were also able of inducing the appearance of a phase transition, but not LPC. Judging from the observed ∆H value observed the effect of edelfosine is stronger than those of PAF and PAPC. The inclusion 33 ACS Paragon Plus Environment

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of the lipid analogs of edelfosine emphasizes the good capacity of this molecule to complex cholesterol, being better suited than a molecule like LPC which is present in the membrane. PAF is also well suited but this molecule is only present in very few cells and in very low concentration. PAPC is not a physiological molecule but edelfosine offers better results. It should be also commented that when either Lα phase or Lo phase are observed in WAXD they show a very broad component with no sharp reflections. The appearance of the reflections at 4.11 and 4.13 Å, on the other hand, are typical of Lβ phases, as previously shown by Luzzati 30. On the other hand, if edelfosine is dispersed alone in water it will form micelles or very small vesicles, as seen by 31P-NMR (Fig. 8). In any case, if edelfosine will form separate particles an additional SAXD pattern would be seen and it is not. Therefore we conclude that the reflections observed in WAXD are due to the formation of a gel phase, also in agreement with DSC and infrared spectroscopy results. The reason why edelfosine may destabilize a liquid ordered phase seems to be its capacity to associate to cholesterol favorably competing with SM. This is the obvious conclusion that can be reached from the appearance of Lβ phases and phase transitions that can be attributed to phases formed by SM and POPC in the case of the ternary system POPC/SM/cholesterol and to predominantly SM with some cholesterol, but not enough to prevent the phase transition, in the case of the simplified SM/cholesterol system. The same explanation can be given to the effects of PAF and PAPC. Why some of these lipids have the capacity to interact with cholesterol more strongly than SM is not clear from the molecular point of view, but it is intriguing why LPC with a free hydroxyl group does not show this capacity for this interaction. It 34 ACS Paragon Plus Environment

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seems that a hydrophobic group at position 2´ of glycerol is necessary, forming an ether bond as edelfosine or an ester one like PAF and PAPC. In addition, it seems that this hydrophobic group must be a short one: a methyl group as it is the case for edelfosine or an ester one like PAF and PAPC. Since edelfosine gives the strongest effect it seems that a methyl group is preferred. On the other hand, with respect to the substitution in position 1´of glycerol is by means of a long hydrophobic chain in all the cases studied here, but with ether bond in the case of edelfosine and PAF and ester bond for PAPC. The capacity of edelfosine to destabilize liquid ordered domains as the rafts found in cells that has been widely illustrated in a number of previous works (see 49 for a review) and the results presented in this paper support the theory that the antitumor drug edelfosine acts through reorganization and modification of lipid rafts in liquid ordered state (Lo) therefore support the consideration of these domains as a promising therapeutic target against cancer 2 and against parasites as Leishmania donovani 50 and Stronglioides venezuelensis 4. PAF and PAPC may also destabilize these liquid ordered domains, however they have been observed to have much lower capacity to induce apoptosis than edelfosine in HL-60 cells 19. This may due to the capacity of the cells to metabolize both compounds unlike edelfosine or to the ability of edelfosine to interact additionally with certain proteins present in the rafts. In the present work, we have employed MD simulations to shed light on the differential behavior of the phospholipids under study in phospholipid/cholesterol membranes. The SM/cholesterol membrane showed a slightly higher value of ACHOL (Table 2). This result could be interpreted as a slightly lower packing of the cholesterol


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molecule in the SM/cholesterol membrane as compared to the rest of lipid/cholesterol bilayers, also in agreement with a lower radial distribution function found for the SM/cholesterol membrane (Fig. 10). Both results point to a lower packing of the cholesterol molecule around the SM molecule as compared with the asymmetric phospholipids. Interaction energies were also calculated as an attempt to characterize the observed experimental results. Thus, edelfosine/cholesterol membrane showed the lowest value of total intermolecular interaction energy, while SM/cholesterol membrane showed the highest value (Fig. 11). These interactions correspond to all the intermolecular interactions: phospholipid-phospholipid, cholesterol-cholesterol, and phospholipid-cholesterol. The origin of the differences in the energy interactions have been found in the phospholipid-cholesterol interaction, mainly in the electrostatic interactions (Fig. 12B). This result would be in favor of a cholesterol preference for an asymmetric phospholipid, e.g., edelfosine, than for SM, facilitating cholesterol partition in the asymmetric phospholipid phase. On the other hand, cholesterol-cholesterol interactions are slightly higher in asymmetric phospholipids than in SM/cholesterol membranes. Therefore, in an asymmetric phospholipid membrane, cholesterol presents more interactions with the phospholipid and with itself than in the SM/cholesterol membrane. This result would be in agreement with the observed higher cholesterol surface area obtained in the SM/cholesterol bilayer (Table 2) and with the radial distribution function from lipid to cholesterol where cholesterol appears at longer distance from SM than the other phospholipids in the XY plane (Fig. 10). Intermolecular hydrogen bonds formation is an important contribution to the electrostatic interaction energy term. Thus, it has been showed a lower capacity of SM to form hydrogen bonds with cholesterol than the rest of the phospholipids (Table 3), in


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agreement with our data of phospholipid-cholesterol electrostatic interaction energies shown in Fig. 12B, supporting a higher interaction of cholesterol with asymmetric phospholipid, e.g., edelfosine, than with SM. All these results point to a higher interaction of cholesterol with asymmetric phospholipids than with SM. Then, in a SM/cholesterol bilayer the addition of asymmetric phospholipids would decrease the SM-cholesterol interactions by promoting the distribution of cholesterol towards the asymmetric phospholipid. However, the overall driving force that would lead to cholesterol segregation in a lipid mixture cannot be explained solely in terms of Coulomb potentials and Lennard-Jones potentials of short range nonbonded interactions. Unfortunately, using PME in Gromacs the longrange interactions cannot be determined. Besides, the entropic contribution to these systems should be taken into account since an important change in entropy could be expected for these systems. An asymmetric phospholipid tends to form micelles and therefore their incorporation in a mixed bilayer would induce curvature and instability of the membrane. The presence of cholesterol would allow the formation of an asymmetric phospholipid-cholesterol tandem which fit well in a lipid bilayer. Thus, cholesterol would buffer the perturbing effect of the presence of an asymmetric phospholipid in a lipid bilayer. Therefore, the interaction energy results presented here cannot fully explain, only by themselves, the behavior of lipid/cholesterol membranes but offer an alternative to address the complex behavior of mixed lipid membranes in terms of interaction energies. In summary, we show in this paper that edelfosine is a potent destabilizer of liquid ordered domains and it is deduced that this must be done because of its capacity to interact with cholesterol more strongly than SM does. PAF and PAPC also show


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capacity to destabilize liquid ordered domains although less potently than edelfosine and in contrast LPC does not show this capacity at all. Conflict of interest There are no conflicts of interest to declare Acknowledgements This work was partially supported by grant 368 from University of Murcia. We thank Lipoid (Steinhausen, Switzerland) for the kind gift of POPC. We thank Monika Schneider for some preliminary experiments.


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16. Guo, W.; Kurze, V.; Huber, T.; Afdhal, N. H.; Beyer, K.; Hamilton, J. A., A solid-state NMR study of phospholipid-cholesterol interactions: sphingomyelin-cholesterol binary systems. Biophys J 2002, 83 (3), 1465-78. 17. Lonnfors, M.; Doux, J. P. F.; Killian, J. A.; HNyholm, T. K. M.; Slotte, J. P., Sterols have higher affinity for sphingomyelin than for phosphatidylcholine bilayers even at equal acyl-chain order. Biophys. J. 2011, 100, 2633-2641. 18. Ausili, A.; Torrecillas, A.; Aranda, F. J.; Mollinedo, F.; Gajate, C.; Corbalan-Garcia, S.; de Godos, A.; Gomez-Fernandez, J. C., Edelfosine is incorporated into rafts and alters their organization. J Phys Chem B 2008, 112 (37), 11643-54. 19. Torrecillas, A.; Aroca-Aguilar, J. D.; Aranda, F. J.; Gajate, C.; Mollinedo, F.; CorbalanGarcia, S.; de Godos, A.; Gomez-Fernandez, J. C., Effects of the anti-neoplastic agent ET-18OCH3 and some analogs on the biophysical properties of model membranes. Int J Pharm 2006, 318 (1-2), 28-40. 20. Lemkul, J. A.; Bevan, D. R., Lipid composition influences the release of Alzheimer's amyloid beta-peptide from membranes. Protein Sci 2011, 20 (9), 1530-45. 21. Koziara, K. B.; Stroet, M.; Malde, A. K.; Mark, A. E., Testing and validation of the Automated Topology Builder (ATB) version 2.0: prediction of hydration free enthalpies. Journal of computer-aided molecular design 2014, 28 (3), 221-33. 22. Martinez, L.; Andrade, R.; Birgin, E. G.; Martinez, J. M., PACKMOL: a package for building initial configurations for molecular dynamics simulations. Journal of computational chemistry 2009, 30 (13), 2157-64. 23. Abraham, M.; Hess, B.; Spoel, D. v. d.; Lindahl, E., GROMACS User Manual Version 5.0.7. www.Gromacs.Org 2015. 24. Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; DiNola, A.; Haak, J. R., Molecular Dynamics with Coupling to an External Bath. J. Chem. Phys. 1984, 81, 3684-3690. 25. Miyamoto, S.; Kollman, P. A., Settle: An Analytical Version of the SHAKE and RATTLE Algorithm for Rigid Water Models. J. Comput. Chem. 1992, 13, 952-962. 26. Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M., LINCS: A Linear Constraint Solver for Molecular Simulations. J. Comput. Chem. 1997, 18, 1463-1472. 27. Parrinello, M.; Rahman, A., Polymorphic Transitions in Single Crystals: A New Molecular Dynamics Method. J. Appl. Phys. 1981, 52, 7182-7190. 28. Schrödinger, L., The PyMOL Molecular Graphics System, Version 1.8;. 2015. 29. Chen, L.; Yu, Z.; Quinn, P. J., The partition of cholesterol between ordered and fluid bilayers of phosphatidylcholine: A synchrotron X-ray diffraction study. Biochimica et Biophysica Acta 2007, 1768, 2873-2881. 30. Luzzati, V., Biological membranes: Physical fact and function Academic Press: London, 1968; Vol. vol. 1, p 71-123. 31. Rappolt, M.; Hickel, A.; Bringezu, F.; Lohner, K., Mechanism of the lamellar/inverse hexagonal phase transition examined by high resolution x-ray diffraction. Biophys J 2003, 84 (5), 3111-22. 32. Cortijo, M.; Alonso, A.; Gomez-Fernandez, J. C.; Chapman, D., Intrinsic protein-lipid interactions. Infrared spectroscopic studies of gramicidin A, bacteriorhodopsin and Ca2+ATPase in biomembranes and reconstituted systems. J Mol Biol 1982, 157 (4), 597-618. 33. Gilbert, D. B.; Tanford, C.; Reynolds, J. A., Cholesterol in aqueous solution: hydrophobicity and self-association. Biochemistry 1975, 14 (2), 444-8. 34. Hofsass, C.; Lindahl, E.; Edholm, O., Molecular dynamics simulations of phospholipid bilayers with cholesterol. Biophys J 2003, 84 (4), 2192-206. 35. Maulik, P. R.; Shipley, G. G., N-palmitoyl sphingomyelin bilayers: structure and interactions with cholesterol and dipalmitoylphosphatidylcholine. Biochemistry 1996, 35 (24), 8025-34. 36. Maulik, P. R.; Atkinson, D.; Shipley, G. G., X-ray scattering of vesicles of N-acyl sphingomyelins. Determination of bilayer thickness. Biophys J 1986, 50 (6), 1071-7. 40 ACS Paragon Plus Environment

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