Membranotropic Effects of Arbidol, a Broad Anti-Viral Molecule, on

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Membranotropic Effects of Arbidol, a Broad Anti-Viral Molecule, on Phospholipid Model Membranes Jose´ Villalaıń* Institute of Molecular and Cellular Biology, UniVersity “Miguel Hernańdez”, E-03206 Elche (Alicante), Spain ReceiVed: March 23, 2010; ReVised Manuscript ReceiVed: May 25, 2010

Arbidol, a broad and potent antiviral molecule, incorporates rapidly into membranes. To gain further insight into the mode of action of Arbidol, since the exact antiviral mechanism of Arbidol is unknown, I examined its interaction and effects on model membranes composed of saturated phospholipids by performing a detailed biophysical study using calorimetry and infrared spectroscopy. Arbidol interacts and modifies the physicochemical properties of the phospholipids in the membrane, having a significant effect on negatively charged phospholipids but a minor one on zwitterionic phospholipids. The data suggest that Arbidol is located at the interface of the membrane, participates in hydrogen bonding either with water or the phospholipid or both, and decreases the hydrogen bonding network of the phospholipids giving place to a phospholipid phase similar to the dehydrated solid one. The significant effects produced on negatively charged phospholipids suggest that the active molecule of Arbidol in the membrane is the protonated one, that is, the positively charged molecule. These data suggest that the potent antiviral effects of Arbidol are mediated at least in part through its membranotropic effects, likely giving place to the formation of perturbed membrane structures. These modifications interfere with proper membrane functioning and should be responsible for its broad antiviral activity. Introduction Arbidol is a potent broad-spectrum antiviral with demonstrated activity against a number of enveloped and nonenveloped viruses,1-7 first marketed in Russia and more recently in China. The chemical name of Arbidol is ethyl-6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-[(phenylthio)methyl]-indole-3-carboxylate hydrochloride monohydrate (molecular formula C22H25BrN2O3S; Figure 1). Arbidol exhibits a broad and potent antiviral activity against a number of viruses including adenovirus, avian coronavirus, avian viruses H5N1 and H9N2, Coxsackie virus B5, hepatitis B virus, hepatitis C virus, infectious bronchitis virus, and influenza viruses, among others.1-7 Those viruses that can be inhibited by Arbidol include enveloped and nonenveloped viruses, RNA and DNA viruses, as well as pH-independent and pH-dependent ones. These differences in viral types emphasize the broad spectrum of Arbidol antiviral activity. It has also been reported that Arbidol has interferon-inducing activity, activated phagocytic activity of macrophages, stimulated humoral and cell-mediated immunity, and low toxicity upon oral administration,4,6,8 but it might be metabolized in vivo to several unknown metabolites.4 Arbidol might prevent adsorption and entry of viruses into cells by inhibiting the fusion of viral lipid membranes with cell membranes.4,5,7 Arbidol incorporates rather easily into lipid membranes,5 but no indication of any specific interaction with particular phospholipid molecules has been found until now. It has been also found that Arbidol increases the stability of influenza virus hemagglutinin as well as with other viral proteins;4 it is then possible that Arbidol, apart from interacting with biomembranes, might interact with viral envelope proteins.4,5 However, the exact antiviral mechanism of Arbidol is still unknown. * To whom correspondence should be addressed. Tel.: +34 966 658 762. Fax: +34 966 658 758. E-mail: [email protected].

The fact that Arbidol principal mode of action is most likely through membrane interaction, it seems reasonable to think that the mode of action of Arbidol might be at least partly attributable to its specific effect on membrane structure as well as specific interactions with particular lipid molecules. Therefore, I have carried out a detailed biophysical study on the interaction of Arbidol with model membranes composed of saturated phospholipids, that is, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-[phospho-racglycerol] (DMPG) 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA), 1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) and 1,2-dielaidoyl-sn-glycero-3-phosphatidylethanolamine (DEPE). To this aim I have used differential scanning calorimetry (DSC) and infrared spectroscopy (IR). The results obtained in this work suggest that the potent antiviral effects of Arbidol might be likely mediated by the formation of perturbed membrane structures which interfere with proper membrane functioning. Materials and Methods Reagents. Arbidol (99.61% purity) was a gift from Dr. G. Rohrabaugh (Good Earth Medicine LLC, WA, U.S.A.).1,2Dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC), 1,2dimyristoyl-sn-glycero-3-[phospho-rac-glycerol] (DMPG), 1,2dielaidoyl-sn-glycero-3-phosphatidylethanolamine (DEPE), 1,2dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA), and 1,2dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) were obtained from Avanti Polar Lipids (Alabaster, AL, U.S.A.). Stock solutions were prepared in chloroform/methanol (2:1) and stored at -20 °C. Water was deionized, twice-distilled and passed through Milli-Q equipment (Millipore Ibe´rica, Madrid, ES) to a resistivity higher than 18 MΩ cm. All other chemicals were commercial samples of the highest purity available (SigmaAldrich, Madrid, ES). Phospholipid concentrations were determined by phosphorus analysis.9

10.1021/jp102619w  2010 American Chemical Society Published on Web 06/07/2010

Effect of Arbidol on Model Membranes

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Figure 1. The chemical structure of Arbidol (ethyl-6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-[(phenylthio)methyl]-indole-3carboxylate-hydrochloride-monohydrate). The unprotonated and protonated forms are depicted in (A) and (B) respectively. Hydrogens have been omitted for clarity.

Vesicle Preparation. Arbidol was dissolved in absolute ethanol followed by dilution in deionized twice-distilled water to a final concentration of 100 mM (water/ethanol 9:1 vol/vol).5 Aliquots of phospholipid in chloroform-methanol (2:1 vol/vol) were placed in test tubes containing different amounts of dried lyophilized Arbidol to obtain different phospholipid/Arbidol molar ratios. After vortexing, the solvents were removed by evaporation under a stream of O2-free nitrogen, and finally, traces of solvents were eliminated under vacuum in the dark for more than three hours. The samples were hydrated in 300 µL of D2O (IR) or H2O (DSC) buffer containing 20 mM HEPES, 100 mM NaCl, 0.1 mM EDTA, pH 7.4, and incubated at 10 °C above the phase transition temperature (Tm) of the phospholipid mixture with intermittent vortexing for 45 min to hydrate the samples and obtain multilamellar vesicles (MLV). The samples were frozen and thawed five times to ensure complete homogenization and maximization of Arbidol/lipid contacts with occasional vortexing. Finally the suspensions were centrifuged at 600 g at 22 °C for 10 min. The pellet was resuspended with 25 µL of D2O buffer (IR) or 25 µL of H2O buffer (DSC) and incubated for 45 min at 10 °C above the Tm of the lipid mixture with occasional vortexing unless stated otherwise. Differential Scanning Calorimetry. Samples containing 2.5 mg of total phospholipid were transferred to 50 ul DSC aluminum and hermetically sealed pans and subjected to DSC analysis in a differential scanning calorimeter Pyris 6 DSC (Perkin-Elmer Instruments, Shelton, U.S.A.). Thermograms were recorded at a constant rate of 1.5 °C/min. After data acquisition, the pans were opened and the phospholipid content was determined. Partial phase diagrams for the phospholipid components were constructed as described previously.10 The onset and completion temperatures of the transition peaks obtained from heating scans defined the solidus and fluidus lines of the diagrams, respectively. To avoid artifacts due to the thermal history of the sample, the first scan was never considered; second and further scans were carried out until a reproducible and reversible pattern was obtained. Data acquisition was performed using the Pyris Software for Thermal Analysis, version 4.0 (Perkin-Elmer Instruments LLC) and Microcal Origin software (Microcal Software Inc., Northampton, MA, U.S.A.) was used for data analysis. Infrared Spectroscopy. Samples containing 1 mg of total phospholipid were placed between two CaF2 windows separated by a 56 µm thick Teflon spacer in a liquid demountable cell (Harrick, Ossining, NY). The spectra were obtained in a Bruker IFS55 FT spectrometer using a deuterated triglycine sulfate detector. Each spectrum was obtained by collecting 256 interferograms with a nominal resolution of 2 cm-1, transformed using triangular apodization and, to average background spectra between sample spectra over the same time period, a sample

shuttle accessory was used to obtain sample and background spectra. The spectrometer was continuously purged with dry air at a dew point of -40 °C in order to remove atmospheric water vapor from the bands of interest. All samples were equilibrated at the lowest temperature for 20 min before acquisition. An external bath circulator, connected to the infrared spectrometer, controlled the sample temperature. Samples were scanned using 2 °C intervals and a 2 min delay between each consecutive scan. Subtraction of buffer spectra taken at the same temperature as the samples was performed interactively using either GRAMS/32 or Spectra-Calc (Galactic Industries, Salem, MA) as described previously.11,12 Frequencies at the center of gravity were measured by taking the top 12 points of each specific band and fitted to a Gaussian band. The criterion used for buffer subtraction in the CdO and amide regions was the removal of the band near 1210 cm-1 and a flat baseline between 1800 and 2100 cm-1. Band-narrowing and curve-fitting were applied as previously reported.13,14 Two-dimensional correlation analysis was carried out using the 2D-Shige program written by Shigeaki Morita and Yukihiro Ozaki (Kwansei-Gakuin University, Japan; http://sci-tech.ksc.kwansei.ac.jp/∼ozaki/ e_2D.htm). To obtain the two-dimensional infrared (2D-IR) maps, heating was used as the perturbation to induce timedependent spectral fluctuations in the infrared spectra, so that to induce the phase transition of the phospholipid membranes and observe any modulation on the Arbidol molecule induced by the phase change. Visualization of the 2D-IR maps was performed using Origin software (Microcal Systems, Boulder, CO). The maximum and minimum intensities for the whole correlation map were found, their values were multiplied by two, and a maximum of twenty contour lines were drawn, so that the number of contour lines reflect their intensities in relation to the main peak. Since the synchronous spectrum is always required for interpretation of intensity and width changes, and our interest aims to find the intermolecular interactions between Arbidol and the phospholipid molecules, I have used throughout this work the synchronous 2D-IR correlation contour maps to analyze the data as it has been done before.15 Results In the first place I will comment on the effects of Arbidol on model membranes composed of zwitterionic phospholipids (i.e., DMPC and DEPE) and in the second place its effects on model membranes composed of negatively charged phospholipids (i.e., DMPG, DMPA and DMPS). These phospholipids were chosen for our study because their thermotropic properties are different but their behavior is well-known. Moreover, their phase transitions and conformational changes are amenable to study and information about any specific interaction between the

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Figure 2. Differential scanning calorimetry heating-scan thermograms (A,B) and partial phase diagrams (C,D) for mixtures of Arbidol plus (A,C) DMPC and (B,D) DEPE. Each sample contained the appropriate amount of Arbidol (molar % of total) as indicated on the curves. Filled circles correspond to the lamellar fluidus line and open circles to the solidus line. Filled squares depict the fluid-lamellar/hexagonal-HII boundary whereas open squares correspond to the fluid-lamellar + hexagonal-HII/hexagonal-HII boundary. G corresponds to a lamellar gel phase, F to a lamellar fluid phase, and HII to the hexagonal-HII phase. Pretransitions have been omitted for clarity.

phospholipid and Arbidol molecules as well as membrane location of Arbidol can be obtained.10,16 I have used DSC and IR with this definite aim in mind. The effect of increasing concentrations of Arbidol on the thermotropic behavior of model membranes composed of DMPC is depicted in Figure 2a. DMPC, when not extensively annealed at low temperatures, exhibits a highly cooperative phase transition that is fully reversible and relatively energetic at about 23 °C, corresponding to the main transition from the Pβ′ gel phase to the liquid-crystalline phase LR, and a less energetic pretransition corresponding to the conversion of the Lβ′ gel phase to the Pβ′ gel phase at about 12-14 °C.17,18 The pretransition of the phospholipid was already shifted to lower temperatures at concentrations as low as 0.99 mol % of Arbidol and was completely abolished at concentrations equal or greater than 1.96 mol % (Figure 2a). On the other hand, Arbidol had a minor effect on the thermotropic behavior of DMPC since increasing concentrations of the molecule did not affect significantly either the enthalpy or the main transition of the phospholipid (Figure 2a). At the maximal concentration of Arbidol used (33.3 mol %), the broad main transition was not abolished (Figure 2a). Aqueous dispersions of DEPE undergo a gel to liquid-crystalline (LβfLR) phase transition in the lamellar phase (about 38 °C) and in addition, a lamellar liquid-crystalline to hexagonal-HII (LRfHII) transition (about 63 °C).19 The effect of increasing concentrations of Arbidol on the thermotropic behavior of model membranes composed of DEPE is shown in Figure 2b. Incorporation of Arbidol into DEPE up to 20 mol % did not

had a significant effect on the main gel to liquid-crystalline phase transition. Higher concentrations of Arbidol gave place to a broadening of the main transition peak and, at the same time, induced the appearance of a small peak at slightly lower temperatures (Figure 2b). Arbidol did not have also a strong effect on the hexagonal-HII phase transition. Up to concentrations of 1.96 mol % of Arbidol, the temperature of the hexagonal-HII phase transition decreased slightly, up to 20 mol % was nearly constant and decreased again at higher molar ratios. Both the main and the hexagonal-HII phase transitions were not abolished at the highest Arbidol molar ratio used (Figure 2b). Using the DSC data, I have constructed partial phase diagrams for the mixtures containing Arbidol and both DMPC and DEPE (Figure 2c,d). The partial phase diagram corresponding to DMPC/Arbidol dispersions (Figure 2c) shows that Arbidol does not present an intense effect on the gel-to-liquid crystalline phase transition of DMPC. Both the temperature of the solidus and fluidus lines decreased slightly as the concentration of Arbidol increased, suggesting that both molecules are miscible in both lamellar gel and lamellar fluid phases (Figure 2c). The partial phase diagram corresponding to DEPE/Arbidol dispersions show that Arbidol does not present a strong effect on the gel-to-liquid crystalline phase transition of DEPE, since the temperature of the solidus and fluidus lines decreased slightly as the concentration of Arbidol increased. However, an immiscibility in the gel phase was observed up to concentrations of 25.92%, indicating the existence of different DEPE/Arbidol domains at high Arbidol



Figure 3. Differential scanning calorimetry heating-scan thermograms (A-C) and partial phase diagrams (D,F,E) for mixtures of Arbidol plus (A,D) DMPC, (B,F) DMPA, and (C,E) DMPS. Each sample contained the appropriate amount of Arbidol (molar % of total) as indicated on the curves. Filled circles correspond to the lamellar fluidus line and open circles to the solidus line. G corresponds to a lamellar gel phase, and F corresponds to a lamellar fluid phase. Pretransitions have been omitted for clarity.

concentrations (Figure 2d). A complex behavior was observed for the phase diagram corresponding to the LR-HII phase transition peak. Up to a concentration of 1.96 mol % of Arbidol, the temperature of the transition decreased, up to 20 mol % remained constant, and decreased again at higher concentrations, indicating the presence of immiscibilities in the hexagonal HII phase (Figure 2d). DMPG, similarly to DMPC, when not extensively annealed at low temperatures, exhibits a highly cooperative phase transition (Pβ′ gel phase f LR liquid-crystalline phase) and a less energetic pretransition (Lβ′ gel phase f Pβ′ gel phase).17,18 Addition of very low concentrations of Arbidol to DMPG abolished the pretransition of the phospholipid; however, and in contrast to DMPC, Arbidol significantly affected the phospholipid main transition of the phospholipid, showing a complex DSC profile at relatively high concentrations of Arbidol (Figure 3a). The main transition peak was already broadened at 4.76 mol %; at 9.09 mol % of Arbidol the broadening of the peak was more apparent, and a shoulder was already noticeable at lower temperatures. The broad transition peak was shifted further to lower temperatures at increasing concentrations of Arbidol; the broad transition was composed of at least two peaks, indicating the presence of different phospholipid domains (Figure 3a). The effect of Arbidol on model membranes composed of DMPA is shown in Figure 3b. DMPA (and DMPS, see below) exhibits a single highly cooperative phase transition that is fully reversible and relatively energetic, corresponding to the main transition from the gel to the liquid-crystalline phase.20,21 No perceptible broadening of the main gel-to-liquid

crystalline transition of DMPA was observed up to a concentration of 1.96 mol % of Arbidol. A slight broadening of the main transition peak was observed at a concentration of 4.76 mol % of Arbidol; higher Arbidol concentrations gave place to a broadened main transition peak and the appearance of at least two more peaks at lower temperatures (Figure 3b). The effect of Arbidol on model membranes composed of DMPS is shown in Figure 3c. In the absence of Arbidol, DMPS presents a single highly cooperative phase transition that corresponds to the chain melting transition from the gel to the liquid-crystalline phases.21 The presence of low concentrations of Arbidol induces a broadening of the main transition (up to 1.96 mol % of Arbidol). Higher concentrations of Arbidol induced the appearance of a complex thermogram, where several peaks at lower temperatures were observed. Similarly to what was observed for DMPC and DEPE dispersions, the broad complex transitions of DMPG, DMPA, and DMPS were not abolished at the maximal concentration of Arbidol used, 33.3 mol % (Figure 3). The phase diagram of DMPG/Arbidol dispersions is more complex than that found for DMPC and DEPE dispersions (Figure 3d). A good mixing behavior between the phospholipid and Arbidol molecules was found in the gel phase up to 20 mol % of Arbidol. Immiscibility in the gel phase was observed at concentrations of 20 mol % and higher, indicating the coexistence of different DMPG-rich domains and Arbidol-rich domains in the gel phase. In the fluid phase an immiscibility was observed for mixtures containing low mol % of Arbidol, until 1.96 mol % of Arbidol (Figure 3d). A decrease in the temperature of the fluidus line was observed at higher concen-

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trations of Arbidol, higher up to a concentration of 20 mol % of Arbidol and lower at higher concentrations (Figure 3d).The fluid phase immiscibility at low concentrations of Arbidol indicated the formation of different enriched domains, both immiscible in the fluid phase. The phase diagram of DMPA/ Arbidol dispersions showed gel phase immiscibility for mixtures up to 1.96 mol % of Arbidol (Figure 3f). Up to a concentration of 20 mol % the solidus line decreased as the concentration of Arbidol increased, but higher concentrations of Arbidol induced the presence of immiscibility in the gel phase, indicating the coexistence of different domains in the gel phase. In the fluid phase and up to 1.96 mol % of Arbidol, a slight temperature increase was observed, indicating the likely presence of two phospholipid domains. At higher concentrations of Arbidol, the temperature of the fluidus line decreased slightly, indicating the absence of any immiscibility in the fluidus phase. In the gel phase and up to concentrations of Arbidol of about 5-7 mol %, the phase diagram of DMPS/Arbidol dispersions showed a relatively good mixing behavior between the phospholipid and Arbidol molecules (Figure 3e). Higher concentrations of Arbidol exhibited a gel phase immiscibility up to the highest concentration of Arbidol used, indicating the formation of different enriched domains. In the fluid phase, fluid phase immiscibility was observed only for DMPS dispersions containing low mol % of Arbidol (until 1.96 mol % of Arbidol) (Figure 3e). At higher Arbidol molar ratios, the temperature of the fluidus line decreased slightly indicating good miscibility in the fluid phase. The coexistence of at least two phases for DMPG, DMPA, and DMPS in the presence of Arbidol at different molar ratios would indicate that one of them would be enriched in Arbidol (phospholipids highly disturbed) whereas the other one would be impoverished in them (phospholipids slightly disturbed). It should be commented that Arbidol presents a high thermal stability, since the effects caused on the phospholipids, as observed by several DSC scans on the same samples, were nearly identical. Although it has been shown that the incorporation of biomolecules in the phospholipid palisade of the membrane can affect not only the phospholipid chain order but also the interchain coupling, a shift in the frequency of the CH2 symmetric stretching band is a reliable index of the phase behavior of a phospholipid dispersion.22 Similarly, the CdO stretching band of membrane phospholipids is sensible to the conformation, hydration state, and hydrogen-bonding interactions in the bilayer interface, giving place to different hydrated and dehydrated states and therefore frequencies.23 To further study the effect of Arbidol on the structural and thermotropic properties of phospholipid membranes, I have studied the effects of Arbidol on DMPC, DEPE, DMPG, DMPA, and DMPS by infrared spectroscopy. Since it is possible by infrared spectroscopy to observe bands from both the phospholipid and Arbidol molecules, I can compare the interaction and effects between both molecules in the same sample at the same time. The temperature dependence of the CH2 symmetric frequency of pure DMPC and DMPC containing 33.3 mol % of Arbidol is shown in Figure 4a. The maximum of the CH2 symmetric frequency of pure DMPC, being sensible to changes in mobility and the conformational disorder of the hydrocarbon chains,22 significantly increased at approximately 23-24 °C, change in frequency which corresponds to the gel-to-liquid crystalline phase transition of the phospholipid. In the presence of Arbidol, the cooperativity of the phase transition of DMPC was not modified, but it was shifted to lower temperatures (to about 20 °C) as it was found by DSC (see above). Significantly, the CH2

Villalaıń stretching frequency increased in the presence of Arbidol at all temperatures, indicating that it induced an increase in the conformational disorder of the hydrocarbon chains of DMPC. The temperature dependence of the frequency at the absorbance maximum of the CdO stretching vibration bands of DMPC and Arbidol for dispersions composed of pure DMPC and DMPC containing 33.3 mol % of Arbidol are shown in Figure 4a, whereas the corresponding infrared spectra are shown in Figure 4c. The maximum of the CdO stretching band of pure DMPC, resulting from the combination of the sn-1 and sn-2 groups in different degrees of hydration, underwent two frequency changes, one corresponding to the pretransition and the other one to the main transition.24,25 In the presence of Arbidol only one transition corresponding to the shifted main transition was observed in accordance with the above commented results. Notably, the CdO stretching band of Arbidol presents a small but evident frequency change (less than 1 cm-1, see insert in Figure 4a) coincident with the change observed for the DMPC molecule, indicating that the Arbidol molecule senses the phospholipid transition and therefore it is inserted in the membrane bilayer. The temperature dependence of the CH2 symmetric frequency of pure DEPE and DEPE containing 33.3 mol % of Arbidol is shown in Figure 4b. Pure DEPE shows a highly cooperative change at approximately 40 °C, corresponding to the gel Lβ to-liquid crystalline LR phase transition of the phospholipid; however, the LR-HII phase transition was not detected.26,27 In the presence of Arbidol, the cooperativity of the Lβ-LR phase transition of DEPE was not modified, but it was shifted to lower temperatures (to about 34-35 °C), in accordance with the DSC measurements. Similarly to DMPC in the presence of Arbidol, the frequency of the CH2 stretching frequency of DEPE increased at all temperatures, indicating that Arbidol induced an overall increase in the conformational disorder of the hydrocarbon chains of DEPE. The temperature dependence of the frequency maximum of the CdO stretching vibration bands of DEPE and Arbidol for dispersions composed of pure DEPE and DEPE containing 33.3 mol % of Arbidol are shown in Figure 4b, whereas the corresponding infrared spectra are depicted in Figure 4d. The maximum of the CdO stretching band of pure DEPE underwent two frequency changes, a decrease in frequency corresponding to the gel-to-liquid crystalline phase transition and an increase in frequency corresponding to the lamellar to hexagonal HII phase transition in accordance with previous studies.26,27 In the presence of Arbidol, the frequency maximum of the CdO stretching band was higher than in its absence, indicating that Arbidol induces a general dehydration of the CdO groups of the phospholipid at all temperatures.22 The maximum of the CdO stretching band of DEPE displayed two frequency changes in the presence of Arbidol, one at approximately 21 °C and the other one at approximately 36 °C. The transition observed at 36 °C coincides with the transition observed by DSC, but the one at 21 °C it is not distinguishable by DSC; on the contrary, the hexagonal phase transition detected at about 55 °C by DSC is not observed by infrared (compare Figures 2b and 4b). The presence of Arbidol produced also remarkable changes in the CdO region of the infrared spectra of DEPE at temperatures between the two transitions commented above, that is, 21 and 36 °C (Figure 4d). The contour of the ester carbonyl band of DEPE was composed of several (at least three) narrow bands that result from changes in the hydrogen bonding interactions of the phospholipid in the interfacial region and in the gel phase.28 In fact, the ester carbonyl band contour of the phospholipid is very



Figure 4. Temperature dependence of the frequencies (A,B) of the CH2 symmetric stretching and the CdO carbonyl stretching bands corresponding to both the phospholipid and Arbidol molecules for samples of DMPC and DEPE in the pure form (9) and in the presence of 33.3 mol % of Arbidol (0). Infrared spectra of the CdO regions of (C) DMPC and (D) DEPE in the pure form and in the presence of 33.3 mol % of Arbidol, as indicated. See text for details. Temperature is indicated on the margin.

similar to the lamellar crystalline Lc2 of phosphatidylethanolamines, but no splitting of the CH2 scissoring band was observed (not shown).28,29 Although it is difficult to evaluate from the spectra, it can be observed that at low temperatures the CdO stretching band of Arbidol is composed of two bands, one at about 1683 cm-1 and another one at about 1687.5 cm-1 (insert, Figure 4d). Increasing the temperature, the lower wavenumber band shifts and combines with the higher wavenumber one (Figure 4b). These data indicate that two different populations of Arbidol exists at low temperatures, most probably Arbidol with different degrees of hydration, either with water or with the phospholipid or both. The shifting lower wavenumber band is the most intense one (data obtained by fitting, not shown for briefness); this Arbidol population should correspond with the one interacting directly with the DEPE molecules since is the one that its frequency shifts at the same temperature of the phospholipid transition temperature (Figure 4b). The temperature dependence of the CH2 symmetric frequency of pure DMPG and DMPG containing 33.3 mol % of Arbidol is shown in Figure 5a. Similarly to DMPC, the maximum of the CH2 symmetric frequency of pure DMPG increased significantly at approximately 23-24 °C, a change in frequency that corresponds to the gel-to-liquid crystalline phase transition of the phospholipid.22 In the presence of Arbidol and in accordance with the DSC data, the phase transition of DMPG was shifted to lower temperatures (about 8-12 °C). The frequency of the CH2 stretching frequency of DMPG did not change in the presence of Arbidol, indicating that Arbidol did

not change the conformational order of the hydrocarbon chains of the phospholipid. The temperature dependence of the frequency at the absorbance maximum of the CdO stretching vibration bands of DMPC and Arbidol for dispersions composed of pure DMPC and DMPC containing 33.3 mol % of Arbidol are shown in Figure 5a, whereas the corresponding infrared spectra are shown in Figure 5d. The maximum of the CdO stretching band of pure DMPG displayed two frequency changes, one corresponding to the pretransition and the other one to the main transition. In the presence of Arbidol, only one transition corresponding to the shifted main transition is barely observed, since the transition is at the edge of the temperature range available. Possibly due to the same reason, the CdO stretching band of Arbidol does not present any frequency change in the temperature range studied (insert in Figure 5a). Interestingly, there is no evidence of the presence of any lamellar crystalline phase of DMPG in the presence of Arbidol.30 The temperature dependence of the CH2 symmetric frequency of pure DMPA and DMPA containing 33.3 mol % of Arbidol is shown in Figure 5b. The maximum of the CH2 symmetric frequency of pure DMPA increased significantly at approximately 49 °C, a change in frequency that corresponds to the gel-to-liquid crystalline phase transition of the phospholipid. In the presence of Arbidol, the phase transition of DMPA was broadened and shifted to lower temperatures (about 45 °C) but the cooperativity and the conformational order of the hydrocarbon chains of the phospholipid were not significantly modified. The temperature dependence of the frequency at the

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Figure 5. Temperature dependence of the frequencies (A-C) of the CH2 symmetric stretching, the COO- carboxyl band of DMPS, and the CdO carbonyl stretching bands corresponding to both the phospholipid and Arbidol molecules for samples of (A) DMPG, (B) DMPA, and (C) DMPS in the pure form (9) and in the presence of 33.3 mol % of Arbidol (0). Infrared spectra of the CdO regions of (D) DMPC, (E) DMPA, and (F) DMPS in the pure form and in the presence of 33.3 mol % of Arbidol, as indicated. See text for details. Temperature is indicated on the margin.

absorbance maximum of the CdO stretching vibration bands of DMPA and Arbidol for dispersions composed of pure DMPA and DMPA containing 33.3 mol % of Arbidol are shown in Figure 5b, whereas the corresponding infrared spectra are shown in Figure 5e. The maximum of the CdO stretching band of pure DMPA showed one frequency change, corresponding to the gel to liquid-crystalline phase transition (about 49-50 °C).24,25 In the presence of Arbidol, two frequency changes were observed, a slight increase in frequency at about 41 °C and a decrease in frequency, not as intense as in pure DMPA, at about 45 °C (Figure 5b). The CdO stretching band of Arbidol was composed of two bands with similar intensity, one at about 1679 cm-1 and another one at about 1687.5 cm-1 (Figure 5e). Increasing the temperature, the lower wavenumber band shifted and combined with the higher wavenumber one; the combined one shifted to even higher wavenumbers at increasing temperatures (Figure 4b). These data would indicate that two different populations of Arbidol with different degrees of hydration exist at low temperatures. The shifting lower wavenumber should correspond to the Arbidol population interacting directly with the DMPA molecules since is the one that its frequency shifted with a transition at about 41 °C, coincidental with the small frequency change observed on the CdO carbonyl band of DMPA (Figure 4b). Both converged populations of Arbidol interact with DMPA since the frequency

of the CdO groups of both phospholipid and Arbidol molecules shifted concomitantly to higher wavenumbers at about 45 °C (Figure 4b). The temperature dependence of the CH2 symmetric frequency of pure DMPS and DMPS containing 33.3 mol % of Arbidol is shown in Figure 5c. The maximum of the CH2 symmetric frequency of pure DMPS increased significantly at approximately 38 °C, a change in frequency that corresponds to the gel-to-liquid crystalline phase transition of the phospholipid. In the presence of Arbidol, the CH2 symmetric band displayed a broad frequency change at lower temperatures, indicative of the complex thermogram observed by DSC (Figures 2c and 5c). Arbidol did not change the conformational order of the hydrocarbon chains of the phospholipid at all temperatures studied. The temperature dependence of the frequency at the absorbance maximum of the CdO stretching vibration bands of DMPS and Arbidol for dispersions composed of pure DMPS and DMPS containing 33.3 mol % of Arbidol are shown in Figure 5c, whereas the corresponding infrared spectra are shown in Figure 5f. The carbonyl stretching region of DMPS contains two bands centered at about 1730 and 1620 cm-1, corresponding to the hydrocarbon chain ester carbonyl group and the headgroup carboxylate, respectively.21 Both infrared bands sense the phase transition,31 since both of them undergo a frequency change at about 37-38 °C, temperature corresponding to the gel-to-liquid



Figure 6. Synchronous 2D-infrared contour maps in the CdO region of samples containing (A) DMPC, (B) DEPE, (C) DMPG, (D) DMPA, and (E) DMPS and Arbidol at a 33.3% molar ratio. The upper spectra represent the diagonal of the synchronous maps. The 2D-IR contour maps have been obtained from the deconvolved original spectra (see text for details).

crystalline phase transition of the pure phospholipid (Figure 5c). For pure DMPS, the frequency of the maximum of the CdO carbonyl band in the gel phase is about 1738 cm-1, whereas in the liquid crystalline phase is about 1730 cm-1; the CdO stretching band contour is characteristic of those two phases.21 In the presence of Arbidol, the infrared CdO carbonyl band of the phospholipid presents a complex pattern between 8-9 °C and 28-30 °C, since several narrow peaks were observed (Figure 5f). Before the first transition at about 8-9 °C, the infrared CdO carbonyl band maximum appeared at about 1737 cm-1, above that temperature different narrow bands were observed, and at 28-30 °C they combined again giving place to a broad band with a maximum at about 1730 cm-1 (Figure 5c). The complex behavior of the CdO band is consistent with the decrease in mobility and the presence of different populations of the ester carbonyl groups.21 The carboxylate band of DMPS in the presence of Arbidol displayed a similar and complex behavior. Whereas the frequency of the carboxylate band for the pure phospholipid changed from about 1620 cm-1 (gel phase) to about 1625 cm-1 (liquid-crystalline phase), in the presence of Arbidol the carboxylate band shifted from about 1623 to 1615 cm-1 at 8-9 °C and then to about 1626 cm-1 at 28-30 °C (Figure 5c). The frequency of 1615 cm-1, observed between 8 and 30 °C, is characteristic of the dehydrated state of DMPS, whereas bands at about 1620-1626 cm-1 are characteristic of the hydrated state.31,32 Significantly, the CdO stretching band of Arbidol behaves in an indistinguishable manner as the DMPS ester carbonyl and carboxylate groups (Figure 5c,f).

Since bands from both the phospholipid and Arbidol molecules are observed in the infrared spectra at the same time, it is possible to correlate the interaction between them by using 2D-IR as it has been done previously.33 The synchronous 2DIR correlation CdO contour maps of the phospholipid dispersions containing 33.3 mol % of Arbidol are presented in Figure 6. For DMPC/Arbidol dispersions (Figure 6a), the most intense autopeaks were located at 1689, 1729, and 1739 cm-1. These frequencies correspond to the CdO group of Arbidol and the hydrated and dehydrated CdO groups of DMPC, respectively. Cross-peaks were observed correlating frequencies 1689/1729 and 1689/1739 cm-1. In the case of DEPE/Arbidol dispersions (Figure 6b), the most intense autopeaks were located at 1720, 1726, 1731, 1738, and 1742 cm-1. These data corroborate that DEPE presents at least five different peaks in the presence of Arbidol (see above). Smaller autopeaks were observed at 1679 and 1690 cm-1 (CdO absorption band of Arbidol). Main crosspeaks were observed correlating frequencies 1679/1731, 1690/ 1720, 1690/1731, and 1690/1742 cm-1. In a similar way to DMPC, the most intense autopeaks found for DMPG/Arbidol dispersions (Figure 6c) were located at 1689, 1729, and 1738 cm-1 (CdO groups of Arbidol and DMPG). Cross-peaks were observed correlating frequencies 1689/1729 and 1689/1738 cm-1. The synchronous correlation map corresponding to DMPA/Arbidol dispersions (Figure 6b) displayed a very intense autopeak at 1731 cm-1. Smaller autopeaks appear at 1721, 1726, 1738, and 1744 cm-1 (DMPA) and 1678 and 1689 cm-1 (Arbidol). Two cross-peaks, correlating frequencies 1678/1738, and 1689/1738 cm-1, were observed. For DMPS/Arbidol

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dispersions several very intense autopeaks corresponding to the COO- and CdO bands of the phospholipid were found at 1615, 1628, 1717, 1725, 1734, and 1741 cm-1, as well as an intense broad cross-peak correlating all of them (Figure 6b). A small autopeak was observed at 1676 cm-1, corresponding to the molecule of Arbidol. Significantly, the 2D-IR spectrum exhibited cross-peaks correlating the 1676 cm-1 peak with all of peaks corresponding to DMPS. In summary, and for all dispersions, cross-peaks were found correlating both phospholipid and Arbidol molecules, confirming the specific and direct interaction between the different populations of Arbidol and the phospholipids at the interfacial level. Discussion Arbidol has been shown to exhibit a broad and potent antiviral activity against a number of different types of virus,2-7,34 displaying a low toxicity upon oral administration in many animal models.4 Arbidol incorporates rapidly into model and biological membranes, but no indication of any specific interaction with particular lipid molecules has been found until now.5 It is supposed that the antiviral mechanism of Arbidol involves inhibition of virus-mediated fusion with the target membrane and therefore blocking virus entry into target cells, but the exact antiviral mechanism of Arbidol is still unknown. The molecule of Arbidol has the capacity of forming hydrogen bonds with the phospholipid molecules, since it has one possible H donor group and four possible H acceptor groups. At the same time, Arbidol is a weak base that could be protonated, particularly at the 3 position, when inserted in the membrane giving place to a positively charged molecule (Figure 1b).4,5 To study the effect, location, and interaction of Arbidol in biological membranes, I have carried out a detailed study by using DSC and IR on the interaction of Arbidol with model membranes composed of synthetic phospholipid molecules, namely DMPC, DMPG, DMPA, DMPS, and DEPE. Arbidol had a minor effect on DMPC since increasing concentrations of the molecule did not affect significantly either the enthalpy or the main transition of the phospholipid, and both molecules were miscible in both lamellar gel and lamellar fluid phases. The frequency of the CH2 stretching frequency of DMPC increased in the presence of Arbidol, indicating that Arbidol induced an overall increase in the conformational disorder of the hydrocarbon chains of the phospholipid. Notably, the CdO stretching band of Arbidol presents a small but evident frequency change coincident with the change observed for the DMPC molecule, indicating that the Arbidol molecule senses the phospholipid transition and therefore it is inserted in the membrane bilayer. DSC and infrared data indicated that the cooperativity of the phase transition is not significantly altered, indicating that Arbidol does not disrupt significantly the packing of the phospholipid; therefore Arbidol would be located at the interface rather than intercalated between the phospholipid molecules. Infrared data supports the existence of an interaction between both Arbidol and DMPC at the membrane interface. Similarly to the effect of Arbidol on DMPE, incorporation of Arbidol into DEPE membranes did not have a significant effect either on the main gel-to-liquid crystalline phase transition or the hexagonal-HII phase transition. Arbidol induced the presence of an immiscibility in the gel phase up to concentrations of 25.92%, indicating the existence of different DEPE/Arbidol domain. The frequency of the CH2 stretching frequency of DEPE increased at all temperatures, indicating that Arbidol induced an overall increase in the conformational disorder of the hydrocarbon chains of DEPE. In the presence of Arbidol, the

Villalaıń frequency maximum of the CdO stretching band is higher than in its absence, indicating that Arbidol induces a general dehydration of the CdO groups of the phospholipid at all temperatures.22 The presence of Arbidol produced also remarkable changes in the CdO region of the infrared spectra of DEPE. The contour of the ester carbonyl band of DEPE is composed of several narrow bands which result from changes in the hydrogen bonding interactions of the phospholipid in the interfacial region and in the gel phase.28 In fact, the ester carbonyl band contour of the phospholipid in the presence of Arbidol was very similar to the lamellar crystalline Lc2 of phosphatidylethanolamines, but no splitting of the CH2 scissoring band was observed (not shown).28,29 These data would indicate that Arbidol interacts with the interfacial region of the bilayer decreasing the hydrogen bonding of the phospholipid CdO groups with the water molecules of the hydration layer producing a phase similar to dehydrated solid one.35 In this way, Arbidol, bearing both H donor and acceptor groups, participates in hydrogen bonding, leaving less water molecules available for interaction with the phospholipid. Interestingly, two different populations of Arbidol were present at low temperatures, most probably Arbidol with different degrees of hydration, either with water or with the phospholipid or both. Saturated phosphatidylethanolamines, due to the strong electrostatic and hydrogen bonding interactions that exist between headgroups, have a tendency to crystallize into dehydrated bilayers.36,37 However, long unsaturated phosphatidylethanolamines, as DEPE, form nonlamellar phases like the hexagonal HII phase but not dehydrated crystalline bilayers. The bilayer to hexagonal HII phase transition can be induced by the introduction of additional conformational disorder into the already disordered liquid crystalline bilayer phase as well as a certain degree of headgroup dehydration.38 As shown above, Arbidol affected but slightly the bilayer to hexagonal HII phase transition of DEPE, indicating that the perturbation of the palisade structure of the phospholipid and the dehydration of the interfacial region induced by Arbidol is not enough to modify the hydrophobic volume and the headgroup area of the DEPE molecule,39 as it has been found for other molecules.40 The 2D-IR confirmed the specific interaction between the two populations of Arbidol and the phospholipid at the membrane interface, indicating that Arbidol is located at the bilayer interface. In contrast to both DMPC and DEPE, Arbidol had a more profound effect on negatively charged phospholipids. Arbidol significantly affected the DMPG main transition, showing a complex DSC profile at relatively high concentrations of Arbidol, indicating the presence of different DMPG-rich domains and Arbidol-rich domains. In contrast to DMPC, the frequency of the CH2 stretching frequency of DMPG did not change in the presence of Arbidol, indicating that Arbidol did not change the conformational order of the hydrocarbon chains of the phospholipid. The phase diagram of DMPA/Arbidol dispersions showed gel phase immiscibility indicating the coexistence of different domains in the gel phase. In the fluid phase, a slight temperature increase was observed, indicating the likely presence of two phospholipid domains. In the presence of Arbidol, two frequency changes are observed, a slight increase in frequency at about 41 °C and a decrease in frequency, not as intense as in pure DMPA, at about 45 °C. These data indicate that two different populations of Arbidol with different degrees of hydration exist at low temperatures. Incorporation of Arbidol into DMPS membranes had a significant effect, since it induced the appearance of a complex thermogram, where several peaks at lower temperatures were observed. Higher concentrations of

Effect of Arbidol on Model Membranes Arbidol exhibited a gel phase immiscibility up to the highest concentration of Arbidol used, indicating the formation of different enriched domains. In the fluid phase, fluid phase immiscibility was observed only for DMPS dispersions containing low mol % of Arbidol (until 1.96 mol % of Arbidol). At higher Arbidol molar ratios, the temperature of the fluidus line decreased slightly indicated good miscibility in the fluid phase. The coexistence of at least two phases in the presence of Arbidol at different molar ratios would indicate that one of them would be enriched in Arbidol (phospholipids highly disturbed) whereas the other one would be impoverished in them (phospholipids slightly disturbed). In the presence of Arbidol, the infrared CdO carbonyl band of the phospholipid presents a complex pattern, since several narrow peaks are observed. The narrowing and frequency change of the CdO and COO- bands are consistent with the decrease in mobility and the presence of different populations of the ester carbonyl groups is characteristic of the dehydrated state of DMPS.21,31,32 Changes in the frequency of the carboxylate band has been attributed to changes in the local environment of the headgroup moiety, possibly because of phase state-induced changes in headgroup conformation and/or orientation.41,42 It is interesting to note that, similarly to DEPE in the presence of Arbidol, no splitting of the CH2 scissoring band of DMPS is observed at any temperature in the presence of Arbidol (not shown for briefness). This data would suggest that, although the CdO carbonyl band contour is reminiscent of the lamellar crystalline Lc phase of DMPS,21 the phase observed between 8 and 30 °C in the presence of Arbidol is not a lamellar crystalline phase. Interestingly, the CdO stretching band of Arbidol exhibits the same behavior of the carbonyl and carboxylate bands of DMPS, since two transitions at about 8 and 30 °C are observed, indicating that the Arbidol molecule interacts directly with the DMPS molecule and sense the same phase transitions DMPS is having. It is therefore obvious that Arbidol affects the interfacial moiety of DMPS by establishing hydrogen bonding with either the interfacial water, leaving less water molecules available for interacting with DMPS, or the phospholipid or both. In all cases, 2D-IR spectra confirmed a direct interaction of Arbidol with negatively charged phospholipids. Arbidol has been shown to exhibit a broad and potent antiviral activity against a number of different types of virus suggesting that Arbidol targets a common step in viral host cell interaction. It is also know that Arbidol incorporates rapidly into membranes, but the exact antiviral mechanism of Arbidol is still unknown. I have shown in this work that Arbidol interacts and modifies the physicochemical properties of both zwitterionic and negatively charged phospholipids. In general, the data obtained in this work suggest that Arbidol is located at the interface of the membrane, participates in hydrogen bonding either with water or the phospholipid or both, and decreases the hydrogen bonding network of the phospholipids giving place to a phospholipid phase similar to the dehydrated solid one. Moreover, the effects produced on negatively charged phospholipid are more significant than on zwitterionic ones, suggesting that the active molecule of Arbidol in the membrane is the protonated one, that is, the positively charged molecule. Given the critical functional and structural importance of both zwitterionic and negatively charged phospholipids in biological membranes as well as the importance of membrane rearrangements needed for viral infection, the demonstrated direct interactions between Arbidol and phospholipids that I have described in this work must be responsible, at least in part, for it broad antiviral activity.

J. Phys. Chem. B, Vol. 114, No. 25, 2010 8553 Abbreviations DEPE DMPA DMPC DMPG DMPS DSC IR

1,2-Dielaidoyl-sn-glycero-3-phosphatidylethanolamine 1,2-Dimyristoyl-sn-glycero-3-phosphatidic acid 1,2-Dimyristoyl-sn-glycero-3-phosphatidylcholine 1,2-Dimyristoyl-sn-glycero-3-[phospho-rac-glycerol] 1,2-Dimyristoyl-sn-glycero-3-phosphatidylserine differential scanning calorimetry infrared spectroscopy

Acknowledgment. I am very grateful to Dr. G. Rohrabaugh (Good Earth Medicine LLC, WA, U.S.A.) for providing me with Arbidol. I thank Dr. F. J. Aranda for his helpful comments and advise on the DSC data, as well as Ana Isabel Go´mez for her technical assistance. I also thank Drs. Morita and Ozaki for making available the 2D-Shige program for 2D-IR. This work was supported by Grant BFU2008-02617-BMC (Ministerio de Ciencia y Tecnologıá, Spain). References and Notes (1) Brooks, M. J.; Sasadeusz, J. J.; Tannock, G. A. Curr. Opin. Pulm. Med. 2004, 10, 197. (2) Leneva, I. A.; Russell, R. J.; Boriskin, Y. S.; Hay, A. J. AntiViral Res. 2009, 81, 132. (3) Zhong, Q.; Yang, Z.; Liu, Y.; Deng, H.; Xiao, H.; Shi, L.; He, J. Arch. Virol. 2009, 154, 601. (4) Boriskin, Y. S.; Leneva, I. A.; Pecheur, E. I.; Polyak, S. J. Curr. Med. Chem. 2008, 15, 997. (5) Pecheur, E. I.; Lavillette, D.; Alcaras, F.; Molle, J.; Boriskin, Y. S.; Roberts, M.; Cosset, F. L.; Polyak, S. J. Biochemistry 2007, 46, 6050. (6) Shi, L.; Xiong, H.; He, J.; Deng, H.; Li, Q.; Zhong, Q.; Hou, W.; Cheng, L.; Xiao, H.; Yang, Z. Arch. Virol. 2007, 152, 1447. (7) Boriskin, Y. S.; Pecheur, E. I.; Polyak, S. J. Virol. J. 2006, 3, 56. (8) Liu, M. Y.; Wang, S.; Yao, W. F.; Wu, H. Z.; Meng, S. N.; Wei, M. J. Clin. Ther. 2009, 31, 784. (9) Bo¨ttcher, C. S. F.; Van Gent, C. M.; Fries, C. Anal. Chim. Acta 1961, 1061, 203. (10) Villalain, J.; Mateo, C. R.; Aranda, F. J.; Shapiro, S.; Micol, V. Arch. Biochem. Biophys. 2001, 390, 128. (11) Contreras, L. M.; Aranda, F. J.; Gavilanes, F.; Gonzalez-Ros, J. M.; Villalain, J. Biochemistry 2001, 40, 3196. (12) Pascual, R.; Moreno, M. R.; Villalain, J. J. Virol. 2005, 79, 5142. (13) Bernabeu, A.; Guillen, J.; Perez-Berna, A. J.; Moreno, M. R.; Villalain, J. Biochim. Biophys. Acta 2007, 1768, 1659. (14) Giudici, M.; Pascual, R.; de la Canal, L.; Pfuller, K.; Pfuller, U.; Villalain, J. Biophys. J. 2003, 85, 971. (15) Lefevre, T.; Arseneault, K.; Pezolet, M. Biopolymers 2004, 73, 705. (16) Mateo, C. R.; Prieto, M.; Micol, V.; Shapiro, S.; Villalain, J. Biochim. Biophys. Acta 2000, 1509, 167. (17) Lewis, R. N.; Zhang, Y. P.; McElhaney, R. N. Biochim. Biophys. Acta 2005, 1668, 203. (18) Prenner, E. J.; Lewis, R. N.; Kondejewski, L. H.; Hodges, R. S.; McElhaney, R. N. Biochim. Biophys. Acta 1999, 1417, 211. (19) Gallay, J.; De Kruijff, B. Eur. J. Biochem. 1984, 142, 105. (20) Lewis, R. N.; McElhaney, R. N. Biophys. J. 2000, 79, 1455. (21) Lewis, R. N.; McElhaney, R. N. Biophys. J. 2000, 79, 2043. (22) Mantsch, H. H.; McElhaney, R. N. Chem. Phys. Lipids 1991, 57, 213. (23) Blume, A.; Hubner, W.; Messner, G. Biochemistry 1988, 27, 8239. (24) Cameron, D. G.; Casal, H. L.; Mantsch, H. H.; Boulanger, Y.; Smith, I. C. Biophys. J. 1981, 35, 1. (25) Cameron, D. G.; Mantsch, H. H. Biochem. Biophys. Res. Commun. 1978, 83, 886. (26) Mantsch, H. H.; Hsi, S. C.; Butler, K. W.; Cameron, D. G. Biochim. Biophys. Acta 1983, 728, 325. (27) Mantsch, H. H.; Martin, A.; Cameron, D. G. Biochemistry 1981, 20, 3138. (28) Lewis, R. N.; McElhaney, R. N. Biophys. J. 1993, 64, 1081. (29) McMullen, T. P.; Lewis, R. N.; McElhaney, R. N. Biochim. Biophys. Acta 1999, 1416, 119. (30) Zhang, Y. P.; Lewis, R. N.; McElhaney, R. N. Biophys. J. 1997, 72, 779. (31) Casal, H. L.; Mantsch, H. H.; Hauser, H. Biochemistry 1987, 26, 4408.

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(32) Casal, H. L.; Martin, A.; Mantsch, H. H.; Paltauf, F.; Hauser, H. Biochemistry 1987, 26, 7395. (33) Bernabeu, A.; Lellys, M. C. A.; Villalain, J. Biochim. Biophys. Acta 2007, 1768, 2409. (34) Deng, H. Y.; Luo, F.; Shi, L. Q.; Zhong, Q.; Liu, Y. J.; Yang, Z. Q. Acta Pharmacol. Sin. 2009, 30, 1015. (35) Salgado, J.; Villalain, J.; Gomez-Fernandez, J. C. Biochim. Biophys. Acta 1995, 1239, 213. (36) Wilkinson, D. A.; Nagle, J. F. Biochemistry 1984, 23, 1538. (37) Katsaras, J.; Jeffrey, K. R.; Yang, D. S.; Epand, R. M. Biochemistry 1993, 32, 10700.

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Membranotropic Effects of Arbidol, a Broad Anti-Viral Molecule, on

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