Ergosterol

Article pubs.acs.org/JPCB

Interaction of Azole Compounds with DOPC and DOPC/Ergosterol Bilayers by Spin Probe EPR Spectroscopy: Implications for Antifungal Activity Francesca Cicogna,† Calogero Pinzino,† Sabrina Castellano,‡ Amalia Porta,‡ Claudia Forte,† and Lucia Calucci*,† †

Istituto di Chimica dei Composti OrganoMetallici, Consiglio Nazionale delle Ricerche − CNR, Area della Ricerca di Pisa, via G. Moruzzi 1, 56124, Pisa, Italy ‡ Dipartimento di Farmacia, Università di Salerno, via Giovanni Paolo II, 84084 Fisciano, Salerno, Italy S Supporting Information *

ABSTRACT: The influence of two antifungal azoles, the newly synthesized compound CPA18 and the commercial drug fluconazole (FLC), on the physical state of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and DOPC/ergosterol bilayers was investigated by spin probe electron paramagnetic resonance (EPR) spectroscopy with the aim of ascertaining if direct interactions with the plasma membrane are implied in the mechanism of action against Candida albicans. 5- and 16-Doxyl-stearic acids (5-DSA and 16-DSA, respectively) were employed to this purpose, and EPR spectra were acquired in the 0 to 40 °C temperature range. Spectral line shapes were analyzed within the theory for slow motion EPR using a microscopically ordered macroscopically disordered model to describe the DOPC multilamellar vesicles and an axially symmetric Brownian model for the spin probe motion. For CPA18 remarkable changes in the molecular organization and dynamics of the phospholipid bilayers were found in the region immediately below the polar head groups, probed by 5-DSA, whereas the bilayer core, probed by 16-DSA, was only slightly affected. On the other hand, no effects of FLC on DOPC bilayers were revealed in the regions examined. Small differences were observed between DOPC and DOPC/ergosterol systems. The direct interactions of CPA18 with model membranes here highlighted may contribute to the observed fungicidal properties against both fluconazole-sensitive and -resistant C. albicans strains.

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INTRODUCTION Azole compounds, such as fluconazole (FLC, Figure 1) and miconazole, are the most widely used antifungal agents.1,2 They

broad-spectrum activities, low toxicity, and different mechanisms of action to be used against life-threatening systemic mycoses.7−9 Recently, the peculiar antifungal activity of a new azole compound, CPA18 (Figure 1),10 was reported by some of us.11 Even though its ability to inhibit Candida albicans Cyp51p is comparable to that of related azole antifungals, CPA18 shows fungicidal activity against both azole-sensitive and azoleresistant strains, and is able to act synergistically with FLC. Moreover, compared with FLC-treated cells, CPA18-treated cells displayed distinctive morphological features, which include marked changes in plasma membrane permeability, alterations in membrane lipid composition, and block of germ tube formation.11 These outcomes suggested for CPA18 an additional antifungal activity, besides inhibition of Cyp51p, which could involve a membrane-active mechanism. Indeed, drug−lipid interactions could induce a modification in the membrane bilayer physicochemical properties, which are directly related to physiological features of cell membranes,

Figure 1. Molecular structure of fluconazole (FLC) and CPA18.

act primarily by inhibition of the activity of 14α-sterol demethylase (Erg11p/Cyp51p).3,4 The resulting depletion of ergosterol and accumulation of 14α-methyl-sterols perturb both the structure of the membrane and its functions, ultimately causing inhibition of cell growth and proliferation. Generally azole antifungals have only fungistatic activity, and their extensive use in clinical practice has led to the development of fungal resistance to these drugs.5,6 These issues have created the need for new and effective antifungal agents exhibiting © 2013 American Chemical Society

Received: July 9, 2013 Revised: August 26, 2013 Published: September 13, 2013 11978

dx.doi.org/10.1021/jp406776x | J. Phys. Chem. B 2013, 117, 11978−11987

The Journal of Physical Chemistry B

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50 mM phosphate buffer (pH 7.8) and vortexing thoroughly. The dispersions were transferred into capillaries which were inserted into quartz sample holders for EPR measurements. All the samples contained 50 mg of DOPC in 50 μL of buffer and the molar ratios between the other components and DOPC were ergosterol/DOPC = 0.5; CPA18/DOPC = 0.1; FLC/ DOPC = 0.1; 5-DSA/DOPC = 0.0005; and 16-DSA/DOPC = 0.0005. EPR Measurements. EPR spectra were recorded using a Varian (Palo Alto, CA, USA) E112 X band spectrometer equipped with a Varian E257 temperature control unit. The temperature was controlled within 0.2 °C. Spectra in the lamellar phase were recorded every 5 °C in the 0 to 40 °C interval upon raising the temperature, waiting 10 min for temperature equilibration. For each spectrum, a microwave power of 2 mW and a modulation amplitude of 0.5 G were employed. The rigid limit spectra (−150 °C) were recorded using a microwave power of 2 mW and a modulation amplitude of 2 G. Line Shape Analysis. The EPR spectral line shapes were analyzed using the nonlinear least-squares (NLLS) fit program developed by Budil et al.,27 based on Freed’s theory of slow motional EPR.28,29 In particular, the microscopically ordered and macroscopically disordered (MOMD) model proposed by Meirovitch et al.30,31 was applied to calculate the spectra of the spin probes in multilamellar dispersions of lipids. The motion of the nitroxide moiety attached to the Cn position of the nDSA spin probe alkyl chain was assumed an axially symmetric Brownian diffusion. The principal components of the diffusion tensor, R∥ and R⊥, represent the rates of rotational diffusion around the axis parallel and perpendicular to the chain segment where the doxyl group is attached; considering an all-trans chain, this segment is parallel to the z axis of the magnetic frame of the nitroxide radical. The order parameter S represents the angular extent of the rotational diffusion of the nitroxide moiety relative to the membrane director and is modeled in the fitting program through the dimensionless coefficient c20 in the ordering potential.27,32−34 Since the spectra were not sensitive to R∥, especially since R∥ > R⊥, this parameter was fixed in the calculations, while S and R⊥ were optimized. The best fit parameters are reported in Tables S1 and S2 of the Supporting Information. The principal components of the g and hyperfine interaction (A) tensors used in the fitting were determined from spectra in the rigid limit. Since differences within the experimental error (0.25 G) were found among the different samples, the following values were employed in all the calculations: gxx = 2.0088, gyy = 2.0064, gzz = 2.0026; Axx = 5.5 G, Ayy = 5.5 G, Azz = 33.9 G for 5-DSA; Axx = 5.2 G, Ayy = 5.2 G, Azz = 32.8 G for 16-DSA. A Lorentzian line width tensor (wxx, wyy, wzz) was employed to account for orientation dependent inhomogeneous broadening,27 with wxx = wyy = wzz and equal to 0.5 and 0.6 G for 16-DSA and 5-DSA spectra, respectively. Thirty orientations were considered. Calculation of CPA18 Properties. LogP and pKa values for CPA18 were calculated using the Advanced Chemistry Development (ACD/Laboratories) Software V11.02 (© 1994− 2013 ACD/Laboratories).

such as membrane permeability and activities of membranebound proteins.12 These interactions are also implied in the partitioning of drugs in membrane phospholipids and in the transport of the drug into the fungal cells and, consequently, in drug uptake and accumulation.13,14 As a consequence, any changes in the status of the lipid phase in the fungal membrane could cause direct cytotoxicity and affect drug resistance. To exploit CPA18 antifungal properties, as well as to develop and design new azole derivatives related to this compound, the understanding of the interactions between this molecule and its targets is of primary importance. To this end, in the present work we applied spin probing electron paramagnetic resonance (EPR) spectroscopy, a technique which is highly sensitive to the fluidity of natural and model membranes,15−19 to the investigation of the nature and extent of CPA18−membrane interactions. In particular, the effect of CPA18 on lipid ordering and dynamics in DOPC and DOPC/ergosterol bilayers was monitored at two different depths, that is, close to the polar head groups and in the bilayer core, exploiting the 5-DSA and 16-DSA spin probes (Figure 2), which bear a doxyl (4,4-

Figure 2. Molecular structure of 5-DSA and 16-DSA spin probes.

dimethyl-N-oxy-oxazolidinyl) group 5 and 16 carbons away from the carboxyl headgroup of stearic acid, respectively. DOPC and DOPC/ergosterol systems were chosen as membrane models since phosphatidylcholine and ergosterol are the main phospholipid and sterol components, respectively, of the C. albicans plasma membrane, in which unsaturated phospholipids constitute a considerable fraction of total phospholipids.12,20−24 Ergosterol not only plays an important role in membrane structure and function, but also influences the pharmacological effects of membrane-active antifungal agents, such as antibiotic polyenes.25,26 FLC was taken as a reference compound as previously done in the screening of the CPA18 antifungal activity.11

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MATERIALS AND METHODS Materials. Fluconazole, ergosterol, 5-DSA and 16-DSA spin probes, and solvents of analytical grade were purchased from Sigma-Aldrich. DOPC was purchased from Avanti Polar Lipids. CPA18 was prepared according to a previously reported procedure.10 Sample Preparation. The DOPC, DOPC-CPA18, DOPCFLC, DOPC/ergosterol, DOPC/ergosterol-CPA18, and DOPC/ergosterol-FLC mixtures were prepared by codissolving the components in chloroform. Spin probe solutions (1 mg/ mL) were prepared in ethanol. After adding the appropriate amount of spin probe solution to the lipid or lipid-azole mixtures and mixing, the solvent was evaporated under a stream of nitrogen gas to form a film in the round-bottom of a test tube. The film was dried under vacuum for 18 h. The lipid multilamellar vesicles were formed by hydrating the films with

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RESULTS Interaction of CPA18 and FLC with DOPC Bilayers. A selection of X-band EPR spectra recorded at different temperatures on multilamellar vesicles (MLV) of DOPC with

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Figure 3. Experimental (black) and calculated (red) EPR spectra of 5-DSA in DOPC bilayers without azole compounds (a) and with CPA18 (b) or FLC (c) at the indicated temperatures.

Figure 4. Experimental (black) and calculated (red) EPR spectra of 16-DSA in DOPC bilayers without azole compounds (a) and with CPA18 (b) or FLC (c) at the indicated temperatures.

and without either CPA18 or FLC is shown in Figures 3 and 4, the former showing the spectra obtained using 5-DSA as spin probe, the latter those using 16-DSA. In the case of DOPC MLVs without drugs, EPR spectra typical of lipid bilayers in a liquid crystalline phase were obtained, with the 5-DSA spectra showing high anisotropy and 16-DSA spectra being almost isotropic because of the rotational mobility gradient typical of lipid bilayers when the nitroxide probe is placed further down the fatty acyl chain toward the center of the bilayer, from a relatively restricted polar head region to a more mobile terminal methyl region.35 Upon raising the temperature, a line shape evolution characteristic of an increasing mobility of the spin probes inside the lipid bilayer

and a decrease in the bilayer order was observed. This is manifested as a decrease in the outer hyperfine splitting (2Azz) for the 5-DSA spectra (Figure 5a) and a decrease in the line widths, without changes in the line positions, for the 16-DSA spectra. In the case of DOPC MLV samples to which CPA18 or FLC were added, the spectra show features similar to those found in the pure DOPC bilayers. However, comparing the spectra of 5DSA in DOPC and in DOPC-CPA18 samples (Figures 3a and 3b) it can be observed that there are remarkable differences in the outer hyperfine splitting (2Azz), which is smaller for the sample containing CPA18 at all the temperatures, the difference increasing with the temperature (Figure 5a). This parameter is 11980



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Figure 5. (a,b) Outer hyperfine splittings (2Azz) measured from the spectra of 5-DSA in the indicated samples. (c,d) Orientational order parameter (S) vs T of 5-DSA in the indicated samples. (e,f) LogR⊥ vs 1000/T for the motion of 5-DSA in the indicated samples; lines represent fits to the Arrhenius equation.

generally considered to be a measure of bilayer fluidity, hence simple inspection of Figure 5a would suggest that CPA18 induces higher fluidity in DOPC bilayers, especially at

temperatures above 10 °C. On the other hand, very similar spectra were obtained for 5-DSA in DOPC MLVs in the absence and in the presence of FLC (Figures 3a and 3c), with 11981



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Figure 6. (a,b) Orientational order parameter (S) vs T of 16-DSA in the indicated samples. (c,d) LogR⊥ vs 1000/T for the motion of 16-DSA in the indicated samples; lines represent fits to the Arrhenius equation.

described in terms of the Arrhenius equation, thus obtaining activation energy values of 30 ± 2 kJ/mol and 20 ± 2 kJ/mol for 5-DSA and 16-DSA, respectively, for all the samples. A comparison between the results obtained for the different samples at the same temperature indicates that for 5-DSA the order parameter in the DOPC bilayer does not change significantly in the presence of FLC but markedly decreases in the presence of CPA18, the difference becoming larger by increasing the temperature (Figure 5c). Correspondingly, the rotational diffusion of 5-DSA remains almost unchanged in the presence of FLC and slows down in the presence of CPA18 (Figure 5e). In the case of 16-DSA, no significant differences were found for S and R⊥ between DOPC and DOPC-FLC samples (Figure 6a,c). On the other hand, with respect to the sole DOPC sample, R⊥ values were systematically slightly higher for the sample containing CPA18, respectively, whereas no significant differences were observed for S (Figure 6a,c). Interaction of CPA18 and FLC with DOPC/Ergosterol Bilayers. A selection of EPR spectra of spin probes in DOPC/ ergosterol MLVs (with ergosterol/DOPC molar ratio equal to 0.5), without and with either one of the two azole compounds, recorded at different temperatures, is reported in Figures 7 and 8 for 5-DSA and 16-DSA, respectively. It must be pointed out

2Azz values within the experimental error (Figure 5a), suggesting a minor influence of FLC on membrane fluidity. As far as 16-DSA spectra are concerned (Figure 4), the line shapes recorded for the different samples at the same temperature show quite subtle differences and simple visual inspection cannot give clear indications on the effects of the two azole compounds on the bilayer core. The calculated spectra obtained by applying a nonlinear least-squares fitting procedure (see Materials and Methods section)27 to the experimental EPR line shapes are also shown in Figures 3 and 4. As it can be seen, the chosen model allowed a good agreement between experimental and calculated spectra to be obtained, the most significant spectral features, such as line positions and widths and line shape evolution with temperature, being well reproduced. The best fitting R⊥ and S parameter values are reported in Figures 5 and 6 (and Tables S1 and S2) for 5-DSA and 16-DSA, respectively. In all cases, S decreases and R⊥ increases regularly with increasing the temperature, with values of S ranging from 0.3 to 0.6 and from 0 to 0.15, for 5-DSA and 16-DSA, respectively, and values of R⊥ ranging from 3.5 × 107 s−1 to 3.3 × 108 s−1 and from 1.5 × 108 s−1 to 5.2 × 108 s−1 for 5-DSA and 16-DSA, respectively. The trends of R⊥ with temperature could be satisfactorily 11982



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Figure 7. Experimental (black) and calculated (red) EPR spectra of 5-DSA in DOPC/ergosterol bilayers without azole compounds (a) and with CPA18 (b) or FLC (c) at the indicated temperatures.

Figure 8. Experimental (black) and calculated (red) EPR spectra of 16-DSA in DOPC/ergosterol bilayers without azole compounds (a) and with CPA18 (b) or FLC (c) at the indicated temperatures.

using the model described above are also shown in Figures 7 and 8 and the corresponding S and R⊥ parameter values are reported in Figures 5 and 6 (and Tables S1 and S2) for 5-DSA and 16-DSA, respectively. For both spin probes, no significant differences were found in the S and R⊥ values between samples differing for the presence of ergosterol, nor for the activation energy values.

that for the samples containing FLC the signal intensity progressively disappeared with increasing the temperature, becoming undetectable above 25 °C. At the moment these findings are not fully understood; we can only postulate a prooxidant action of FLC on ergosterol, activated by the temperature, leading to radical species scavenged by the doxyl groups. No other noticeable differences could be observed in the spectra recorded in the same experimental conditions on samples differing only for the presence of ergosterol. This was confirmed by the parameters determined from the spectral analyses. For instance, the 2Azz values measured in 5-DSA spectra were within the experimental error, as it can be seen by comparing Figures 5a and 5b. The best-fit spectra obtained

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DISCUSSION

The distinct outstanding properties of CPA18, compared to related azole compounds FLC and miconazole,11 prompted us to obtain further information on its mechanism of action. Morphological differences of C. albicans cells induced by 11983



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and perpendicular, respectively, to the preferred orientation of the probe acyl chain. Although this is a simple approximation to the complex internal modes of motion of the chain as well as the overall motion,47 this model is commonly adopted for spin probes in lipid bilayers. The order parameter S is a measure of the angular extent of the reorientation of the rotational diffusion axis relative to the membrane director. The smaller the S value, the less restricted is the motion, which usually means that laterally the lipid molecules surrounding the probe are packed less tightly. However, it must be pointed out that Xband EPR spectra of macroscopically unaligned vesicles intrinsically have low resolution, since the inhomogeneous broadening resulting from the superposition of spectra from all orientations tends to mask the homogeneous broadening related to the molecular dynamics. As a consequence, the fitting of the spectra tends to be insensitive to the chosen model and, within a given model, to some of the fitting parameters.32,48 In our case, the spectral line shapes were not sensitive to the rate of rotational diffusion around the long chain axis (R∥), as found by other authors,32−34 so that, after initial estimates, R∥ was fixed in the fittings (values are reported in Tables S1 and S2). The spectra could be well reproduced using the rotational diffusion coefficient R⊥ and the order parameter S as fitting parameters, except for the 5-DSA spectra recorded above 20 °C on the DOPC-CPA18 and, especially, the DOPC/ergosterolCPA18 samples (see Figures 3b and 7b). In these cases, in order to improve the reproduction of the spectra, we introduced additional fitting parameters, but with unsatisfactory results. In particular, the introduction of a diffusion tilt angle (with values of 30 ± 5°), that is, an angle between the z axes of the magnetic and molecular frames, which could take into account the presence of gauche conformations and/or kinks in the probe acyl chain, allowed a better reproduction of the line shape curvature at high field but did not improve the spectral features at low field, as exemplified in Figures S1 and S2. Moreover, the fittings performed in this way gave lower values for R⊥ and higher values for S, rendering difficult a direct comparison with the parameters obtained for the spectra of 5DSA in the other samples. A nonsubstantial improvement to the line shape reproduction was obtained by introducing a nonsymmetric order parameter, representing the nonaxiality of the preferential orientation of the probe relative to the membrane director. Notably, a better reproduction was possible only by introducing a superposition of spectral components corresponding to spin probes with different chain conformation equilibrium or order, both modeled by the S parameter, and/or dynamic properties modeled through the rotational diffusion coefficient R⊥. Indeed, the progressively worse agreement between experimental and calculated spectra observed by increasing the temperature, clearly visible in Figures 3b and 7b, could derive from a progressive increase of disorder in the DOPC bilayers, including both an increase in the amplitude of the rotational diffusion motion and an increase of the gauche conformation population in the chain, which cannot anymore be represented by a unique “average” spectral component. However, the fitting of the experimental spectra using more than one component introduced uncertainties regarding the uniqueness of the fits. For this reason, we chose to use the simpler fits obtained assuming just one component with effective parameters for the motion rate and amplitude, R⊥ and S, respectively, keeping in mind that, considering the assumptions made, the spectral analyses should be used to

CPA18 with respect to FLC, its selective effect on membrane integrity, as well as the selective fungicidal activity against both fluconazole-susceptible and -resistant strains of C. albicans suggested a membrane-active mechanism. As a matter of fact, besides the well-known polyenes as amphotericin B, several compounds exert a considerable fungicidal activity by disturbing the membrane structure.36−40 Moreover, lipids, in addition to being the structural and metabolic components of yeast cells, also play an important role in the acquisition of drug resistance.12,24,41,42 Specifically, the importance of membrane fluidity and asymmetry in affecting resistance to azoles in C. albicans has been reported.43 On the basis of these considerations, we decided to investigate the structural modifications induced by CPA18 on membrane bilayers by means of EPR spectroscopy taking FLC as reference compound. It must be noted that FLC-membrane interactions have been already investigated by other authors, although using different techniques, drug concentration, and saturated, instead of unsaturated, phospholipids.44,45 The EPR spectra obtained on DOPC and DOPC/ergosterol samples, with and without the antifungal azoles CPA18 and FLC, in the 0 to 40 °C temperature range are typical of spin probes in a liquid crystalline phase. Indeed, on the basis of the DOPC/water phase diagram,46 the DOPC bilayers used as model membrane in this study should be in a lamellar Lα phase, representative of a fluid membrane environment, throughout the whole temperature range investigated. As a matter of fact, EPR line shapes reflect the motional and structural properties of the environment in the proximity of the unpaired electron. nDSA spin probes in vesicles experience both internal motions of the chain segments around the C−C bonds and overall reorientation of the molecules within the bilayers, which is normally assumed to be an anisotropic orientational diffusion about the long molecular axis. Spectral line shapes reflecting the rotational mobility gradient typical of lipid bilayers were obtained for 5-DSA and 16-DSA spin probes at the same temperature for all the samples. The location of the doxyl groups of 5- and 16-DSA in the bilayer, i.e., in the upper part of the alkyl chains and deep inside the bilayer, respectively, is supported by the values of the isotropic nitrogen hyperfine coupling constant, aN = 1/3(Axx + Ayy + Azz), of the two spin probes and by the Azz values determined from the rigid limit spectra. In fact, for all samples, 5-DSA has aN = 14.9 G and Azz = 33.9 G, whereas 16-DSA has aN = 14.4 G and Azz = 32.8 G, in agreement with the decreased polarity of the bilayer going from the head groups to the center.19 Moreover, the spectral line shape evolution with increasing the temperature was characteristic of the progressive increase in rotational diffusion rate and concomitant decrease in orientational order for the membrane lipids. Quantitative information on the lipid organization and dynamics in the DOPC bilayers and on the effects of the two azole antifungals on these properties was obtained by fitting the line shapes of the experimental EPR spectra. Since in MLV’s the lipid chains are uniformly oriented within a local bilayer region, but the directions of alignment (i.e., directors which are perpendicular to the bilayer plane) are randomly distributed, a MOMD model30,31 was applied. The EPR spectrum calculated with this model results from the weighted sum of EPR spectra from all orientations. The mobility of the spin probe incorporated into the lipid bilayer was instead modeled as axially symmetric Brownian rotational diffusion. In this model, R∥ and R⊥ represent the rotational motion about an axis parallel 11984



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the phospholipids by means of electrostatic interactions, although the partition coefficients of the ionized species in the lipid bilayers are lower than those of the neutral ones.52−55 Moreover, for bases it has been reported that the positively charged forms bind to the bilayer closer to the water interface with respect to the neutral species.53,52 In the case of CPA18 here investigated, we can speculate that it binds to the DOPC bilayer with the long molecular axis approximately parallel to the fatty acyl chains and the imidazole moiety interacting with the polar head groups, the presence of a significant fraction of positively charged molecules favoring electrostatic interactions with the phosphate groups. However, the insertion of CPA18 in the bilayer is not expected to be deep enough to affect the properties of the DOPC bilayer in the core region probed by 16-DSA. On the other hand, FLC, being quite soluble in water and having a branched structure, probably resides at the surface of the DOPC bilayer. The induction of a looser packing of phospholipids in the membrane bilayer can act synergistically with the depletion of ergosterol and accumulation of 14αmethyl-sterols to induce the severe damages and increased permeability observed in the C. albicans cell membranes after treatment with CPA18.11 Moreover, as mentioned above, the lipid-CPA18 interactions could also account for its antifungal action on FLC-resistant strains of C. albicans. As it can be observed from the EPR spectral line shape and best fit parameter comparison (Figures 3−8), the addition of ergosterol causes minor changes in the DOPC bilayer properties as probed by 5-DSA and 16-DSA. Although ergosterol, as other sterols, interacts with phospholipids influencing the fluidity of membranes, it is well-known that ergosterol has much weaker effects on packing and dynamics of unsaturated than saturated phospholipids, these effects being also dependent on the ergosterol/phospholipid molar ratio.56−58 In particular, previously reported spin probe EPR studies showed comparable spectral features for DOPC and DOPC/ergosterol systems having an ergosterol/DOPC molar ratio of about 0.5.59 On this basis, our results are not surprising. In samples containing CPA18, the presence of ergosterol seems to influence the disordering effects of the azole compound on the DOPC bilayer region probed by 5-DSA, as suggested by the spectral line shapes at higher temperatures. Although no specific interactions between CPA18 and ergosterol can be invoked, these findings indicate that the changes in membrane properties induced by CPA18-DOPC interactions are amplified in the presence of ergosterol. On the contrary, in samples containing FLC, the presence of ergosterol did not change the effect of the drug on the DOPC bilayer.

interpret trends rather than to yield parameters with absolute significance. For all the samples, the best fit values of R⊥ and S were compatible with the liquid crystalline phase formed by DOPC in the concentration and temperature conditions used in the present study. No discontinuity was found in the trends of these parameters with temperature, indicating the absence of phase transitions possibly induced by the presence of the azole compounds. Furthermore, the trends of R⊥ with temperature could be well described in terms of the Arrhenius equation, allowing the activation energy for the reorientational motion to be determined; in general, higher activation energy values were found for 5-DSA with respect to 16-DSA due to the higher sensitivity to temperature of the lipid chain fragments close to the polar head groups.49,50 Our data clearly indicated a marked effect of CPA18 on the physicochemical properties of the model membrane. Indeed, the presence of this azole compound in the DOPC bilayers resulted in a decrease of both R⊥ and S values for 5-DSA and in a slight increase of R⊥ for 16-DSA. Therefore, we can infer that CPA18 interacts with the DOPC bilayers loosening the phospholipid packing in the hydrocarbon phase immediately below the bilayer surface, thus allowing the number of gauche conformers of the spin probe chain and, consequently, the amplitude of the rotational diffusion motion, monitored by the order parameter S, to increase. On the other hand, smaller R⊥ values correspond to a slowing-down of the spin probe reorientational motions. In the bilayer central region, where the packing of DOPC molecules results in an almost isotropic environment, the effect of CPA18 is seen as a slightly faster wobbling of the spin probe. Contrarily to CPA18, DOPC bilayer properties, as probed by 5- and 16-DSA, were not affected by FLC, as indicated by comparing both line shapes and fitting parameter values for DOPC and DOPC-FLC samples (Figures 3−6). These outcomes strongly suggest that direct interaction with the plasma membrane is much more important for the antifungal action of CPA18 than for that of FLC and could, at least in part, explain the remarkable fungicidal properties of this new azole derivative. The differences observed between CPA18 and FLC could be due to their different polarity, hydrophobicity, and steric arrangement. The CPA18 imidazole moiety has higher basicity than the FLC triazole, the pKa values for the conjugated acids being 6.88 ± 0.20 (calculated) and 2.03 (from ref 51), respectively. Therefore, at the investigated pH of 7.8 FLC is essentially in the neutral form, whereas about 11% of the CPA18 molecules are expected to be positively charged52 and could electrostatically interact with the DOPC polar head groups. On the other hand, the comparison of the LogP value estimated for CPA18 (5.21 ± 0.39) with that reported for FLC51 (LogP = 0.5) points to a much higher hydrophobicity of CPA18, which could favor the insertion of this molecule in the DOPC bilayer by hydrophobic interactions. To this respect, the presence of two phenyl rings and the elongated shape of the biphenyl moiety of CPA18 could be of help. However, the LogP parameter, determined in octanol−water, is not sufficient to account for the partition of ionizable molecules in lipid bilayers consisting of amphipathic groups with strong electrostatic interactions, especially at pH values where charged species are present. As a matter of fact, it has been shown that in the phospholipids−water system the partitioning of the ionized species is significantly enhanced over that in the octanol−water system because these species can associate with

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CONCLUSIONS EPR spectroscopy was applied to multilamellar vesicles of DOPC, used as model membrane bilayers, to obtain information on the effect of the azole antifungals CPA18 and FLC on the orientational order and lipid dynamics in fungal cell membranes with the aim of explaining the superior in vitro antimycotic properties of the first agent. Our data indicate that CPA18 induces marked modifications on the microscopic properties of DOPC bilayers, slightly amplified in ergosterolcontaining systems. The major changes were observed in the bilayer region close to the polar head groups and could be correlated to the presence of the imidazole group, the higher lipophilicity, and the quite elongated shape of CPA18. On the other hand, interactions between FLC and DOPC bilayers were found to be modest. These outcomes strongly suggest for 11985



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CPA18, beyond inhibition of 14α-sterol demethylase, a significant direct interaction with the plasma membrane as additional activity within C. albicans cells. The synergistic effect of these actions could account for the fungicidal activity of CPA18 against both fluconazole-sensitive and fluconazoleresistant strains. Overall, these findings highlighted the implication of changes of membrane bilayer physicochemical characteristics in azole anti-Candida activity and could assist in the future development of new and potent antifungal compounds.

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ASSOCIATED CONTENT

S Supporting Information *

Parameters used in the EPR line shape analysis, and examples of the influence of the diffusion tilt angle on the EPR spectra reproduction. This material is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

Corresponding Author

*Mailing address: Istituto di Chimica dei Composti OrganoMetallici, Consiglio Nazionale delle Ricerche − CNR, Area della Ricerca di Pisa, via G. Moruzzi 1, 56124, Pisa, Italy. Phone: +39-050-3152517; Fax: +39-050-3152555; e-mail: lucia. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from Ministero dell’Università e della Ricerca Scientifica e Tecnologica PRIN 2008 (20088L3BP3 to L.C., F.C, S.C., and A.P.). Dr. M. Cristina Sgherri is kindly acknowledged for technical assistance.

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ABBREVIATIONS: DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine; doxyl: 4,4dimethyl-N-oxy-oxazolidinyl; 5-DSA: 5-doxyl-stearic acid; 16DSA: 16-doxyl-stearic acid; CPA18: 1-(1-(biphenyl-4-yl)-3-(4fluorophenyl)propan-2-yl)-1H-imidazole; FLC: fluconazole; EPR: electron paramagnetic resonance; MOMD: microscopically ordered macroscopically disordered; MLV: multilamellar vesicles

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REFERENCES

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